Cellulase compositions and methods of use

ABSTRACT

The subject invention relates to novel compositions of neutral and/or alkaline cellulase and methods for obtaining neutral and/or alkaline cellulase compositions from Chrysosporium cultures, in particular Chrysosporium lucknowense. This invention also provides mutants and methods of generating mutants of Chrysosporium capable of producing neutral and/or alkaline cellulose. This invention also relates to the genes encoding the enzymes comprising the neutral and/or alkaline cellulase composition. In addition, this invention provides methods of culturing Chrysosporium to produce neutral and/or alkaline cellulases. The neutral and/or alkaline cellulase compositions of the subject invention can be used in a variety of processes including stone washing of clothing, detergent processes, deinking and biobleaching of paper &amp; pulp and treatment of waste streams.

FIELD OF THE INVENTION

This invention relates to neutral and/or alkaline cellulases and novelmethods for producing the same. More specifically this invention relatesto cellulases produced by fungi of the genus Chrysosporium, andparticular strains ok Chrysosporium lucknowense. This invention alsorelates to industrial uses for the neutral or alkaline cellulases andcompositions comprising the same.

BACKGROUND OF THE INVENTION

Clothing made from cellulosic fabrics such as cotton, linen, hemp,ramie, cupro, lyocell, newcell, rayon, polynosics, are very popular. Ofparticular interest are clothing items such as jeans made fromindigo-dyed denim fabrics made of cotton or cotton blends. Such clothingitems are typically sewn from sized and cut cloth and tend to be stiffdue to the presence of sizing compositions. In other cases the fibers orrolls of fabric are treated with enzymes prior to sewing the finalgarment. After a period of wear, the clothing items can develop acertain degree of softness, an overall reduction of shade as well aslocalized areas of color variation. Additionally, after repeated washingthe garment continues to provide a more comfortable fit, a softer feeland a worn appearance. In recent years such comfort, feel and appearancehave become increasingly popular.

The most widespread methods for producing this comfort, feel and lookinvolve washing, of clothing items with cellulases in large washingmachines with pumice stones or other abrasives. The pumice helps softenthe fabric and helps to provide the faded surface similar to thatproduced by the extended wear of the fabric. However, the use of pumicehas some disadvantages. For example, the pumice must be manually removedfrom processed clothing items because it tends to accumulate in pockets,on interior surfaces, in creases, and in folds. Also, the pumice stonescan cause overload damage to electric motors of stone washing machines,and clog machine drainage passages and drain lines. These processing andequipment problems can add significantly to the cost of doing businessand to the purchase price of the goods.

In view of the problems of using pumice, alternative methods to usingpumice or other abrasives in the stone washing process have been sought.One alternative involves the use of enzyme treatments which break downthe cellulose in fabrics (Geller U.S. Pat. No. 4,951,366; Olson U.S.Pat. Nos. 4,832,864, 4,912,056, Olson et al. U.S. Pat. Nos. 5,006,126,5,122,159 and 5,213,581, Christner et al. U.S. Pat. No. 4,943,530, Boeghet al. U.S. Pat. No. 4,788,682). Methods for treating cellulosecontaining fabrics with hydrolytic enzymes, such as cellulases, areknown in the art to improve the softness or feel of such fabrics (NovoBrochure Cellulase SP 227; Novo Brochure Celluzyme; Murata U.S. Pat. No.4,443,355; Parslow U.S. Pat. No. 4,661,289; Tai U.S. Pat. No. 4,479,881;Barbesgaard U.S. Pat. No. 4,435,307; Browning UK Patent No. 1,368,599).

Cellulases are known in the art as enzyme systems that hydrolyzecellulose (β-1,4-glucan linkages), thereby resulting in the formation ofglucose, cellobiose, cellooligosaccharides, and the like. Cellulasecompositions are comprised of several different enzyme components,including those identified as exocellobiohydrolases, endoglucanases, andβ-glucosidases. Moreover, these classes of enzymes can be furtherseparated into individual isoenzymes.

The complete cellulase system is required to efficiently convertcrystalline cellulose to glucose. Generally, if total hydrolysis of acellulose substrate is needed, the cellulase mixture should containβ-glucosidases and cellobiohydrolases, as well as endoglucanases.Endoglucanases catalyze random hydrolysis of β-1,4-glycosidic bondsbetween glucose units of cellulose polymers. Such components hydrolyzesoluble cellulose derivatives such as carboxymethylcellulose, therebyreducing the viscosity of such solutions. Such enzyme components act oninternal regions of the polymer, resulting in a rapid decrease inaverage polymer chain length together with a slow increase in the numberof reducing ends. The rapid decrease in average chain length of thecellulose polymer is evidenced by the decrease in viscosity of acellulose solution.

The substrate specificity and mode of action of the different cellulasesvaries among strains of organisms that produce cellulases. For example,the currently accepted mechanism of cellulase action in cellulase fromthe fungus Trichoderma reesei is that endoglucanase activity first breakinternal β-1,4-glucosidic bonds in regions of low crystallinity of thecellulose (Ruohnen L., et al. In: "Proceedings of the Second TricelSymposium on Trichoderma Reesei Cellulases and Other Hydrolases", (ed.by P. Sudminen and T. Reinkainen.,) Foundation for Biotechnology andIndustrial Fermentation Research 8; (1993):87-96) The cellobiohydrolaseactivity binds preferentially to the crystalline regions of thenon-reducing end of the cellulose to release cellobiose as the primaryproduct. β-Glucosidase or cellobiase activities then act oncellooligosaccharides, e.g., cellobiose, to give glucose as the soleproduct.

Cellulases are produced in fungi, bacteria, and other microbes. Fungitypically produce a complete cellulase system capable of degradingcrystalline forms of cellulose. For example, Trichoderma reesei producesand secreates all of the enzyme activities needed for efficientbreakdown of crystalline cellulose, namely endo-1,4-β-D-glucanases,cellobiohydrolases (exo-1,4-β-D-glucanases), and 1,4-β-D-glucanases, orβ-glucosidases. Fungal cellulases have an added advantage in thatcellulases in fungi can readily be produced in large quantities viafermentation procedures.

Cellulases, or the components thereof, are known in the art to be usefulin a variety of industrial textile applications in addition to the stonewashing process. For example, cellulases are used in detergentcompositions, either for the purpose of enhancing the cleaning abilityof the composition or as a softening agent. When so used, the cellulasewill degrade a portion of the cellulosic material, e.g., cotton fabric,in the wash, which facilitates the cleaning and/or softening of thefabric. The endoglucanase components of fungal cellulases have also beenused for the purposes of enhancing the cleaning ability of detergentcompositions, for use as a softening agent, and for use in improving thefeel of cotton fabrics, and the like. However, there is a problem withusing the cellulase derived from Trichoderma spp. and especiallyTrichoderma longibrachiatum in detergent compositions. Generally, suchcomponents have their highest activity at acid pHs whereas most laundrydetergent compositions are formulated for use at neutral or alkalineconditions.

Other textile applications in which cellulases have been used includesoftening (Browning, UK Patent No. 1,368,599, Parslow, U.S. Pat. No.4,661,289, Tai U.S. Pat. No. 4,479,881 and Barbesgaard, U.S. Pat. No.4,435,307), defibrillation (Gintis, D. Mead, E. J., Textile ResearchJournal, 29, 1959; Cooke, W. D., Journal Of The Textile ResearchInstitute, 74, 3, 1983; Boegh, European Patent Application No. 0 220016). Cellulases have also been used in combination with a polymericagent in a process for providing localized variation in the colordensity of fibers. (WO/94/19528 and WP/94/1529).

Cellulases are classified in the garment and textile industry accordingto their pH range of operation. Acid cellulases typically have theirpeak activity at pH values of about 4.0 to 5.5 and less, neutralcellulases at about pH 5.5 to 7.5, and alkaline cellulases at about pH7.5 to 11.0. Some enzyme compositions may have broader ranges ofoperation. For example, the neutral/alkaline cellulases may operate atacid, neutral and alkaline pH's at between about 40° C. to 60° C.

Acid, neutral and alkaline cellulases are typically used in the "stonewash" treatment of denim jeans, with or without surfactants, buffers,detergents, anti-redeposition agents, softening agents, pumice stones orother abrasives, bleaching agents, such as optical bleaching agents,enzymes, or other means. If the cellulase composition is not formulatedand/or pre-buffered then for acid cellulases, the pH is typicallyadjusted to between pH 4.5-5.5, with for example, a sodium citrate andcitric acid buffer, and for neutral or alkaline cellulases between5.5-7.5 with, for example, a monosodium and disodium phosphate buffer.Neutral and alkaline cellulases are typically used as additives tolaundry detergents where the pH of operation may range from about pH 7.0to 11.5. In stone wash applications typical acid cellulases generallyprovide greater backstaining or redeposition of the indigo dye andgreater strength loss of the fabric while the typical neutral andalkaline cellulases generally provide less abrasion, lower backstainingor redeposition and less strength loss of the fabric.

The neutral/alkaline cellulases are the most preferred type ofcellulases for the stonewash industry because they cause lower levels ofbackstaining or redeposition and lower strength loss than acidcellulases (ie; from Trichoderma sp.). Furthermore, neutral/alkalinecellulases, unlike their acid counterparts, operate at a much wider pHrange and are able to maintain better relative wash performance within awider pH range (pH 5.0-pH 8.0) in the stone washing industry. Therefore,neutral/alkaline cellulases provide several advantages. First, theincoming feed water in wet processing facilities is typically withinthis pH range lessening the need for as precise pH control as comparedto acid cellulases. This makes the stonewashing process more tolerant tooperator pH error or neglect leaving the overall procedure moreforgiving than procedures using acid cellulases. Secondly, it is knownthat denim fabrics are alkaline in nature owing to the fact that thedyeing process utilities caustic soda. Simply washing denim releasesthis caustic into the wash water and the pH of the wash water generallyrises. The alkalinity may overcome the bath buffers, but the effect ofincreased pH is less severe on neutral/alkaline cellulases compared toacid cellulases because neutral/alkaline cellulases operate not only athigher pH, but also over a wider pH range.

The wide spectrum of industrial uses for cellulases or the components ofcellulases, especially alkaline and/or neutral cellulases, establishes aclear need for cellulases that are operative at neutral and/or alkalinepH. The present invention provides a procedure for producingneutral/alkaline cellulases having enzymatic activity at neutral and/oralkaline pH's and compositions comprising the same.

SUMMARY OF THE INVENTION

This invention relates, in general, to neutral and/or alkalinecellulases and novel methods for producing the same. More specifically,the subject invention provides a method for producing cellulasecompositions from fungi of the genus Chrysosporium, and particularChrysosporium lucknowense, wherein the cellulase compositions haveenzymatic activity at neutral and/or alkaline pH's. Industrialapplications for the cellulase composition are also provided.

One embodiment of this invention relates to isolated and purifiedcultures of wild type and mutant fungi of the genus Chrysosporiumcapable of producing neutral and/or alkaline cellulase compositions, inparticular to the strain Chrysosporium lucknowense--GARG 27K and mutantsthereof.

Yet another embodiment of this invention provides culturing conditionsfor producing neutral or alkaline cellulases from fungi of the genusChrysosporium.

In a further embodiment, this invention provides methods to producing aneutral and/or alkaline cellulase composition through recombinanttechnology from fungi of the genus Chrysosporium.

In yet a further embodiment of this invention methods for generating andculturing mutant strains of the fungi Chrysosporium capable of producingneutral and/or alkaline cellulase are provided.

Another embodiment of this invention relates to the nucleic acidsequences encoding the enzymes of the cellulases compositions producedby Chrysosporium or genetically modified strains of Chrysosporium.

Another embodiment relates to the purified and isolated enzymes of thecellulase compositions produced by Chrysosporium or genetically modifiedstrains of Chrysosporium.

In yet another embodiment of this invention methods of use are providedfor alkaline and/or neutral cellulases produced by Chrysosporium intextile applications, such as softening, bleaching and stone washingprocedures, garment dyeing applications, defibrillation, orbiopolishing.

Another embodiment of this invention relates to detergent compositionscomprising Chrysosporium cellulase in detergent preparations.

Another embodiment of this invention is to provide methods of use forthe cellulase compositions in the saccharification of lignocellulosebiomass from agriculture, forest products, municipal solid waste, andother sources.

Yet other embodiments of this invention involve the use of the cellulasecompositions for production of fuels and other chemicals for thebiobleaching of wood pulp, and for de-inking of recycled print paper.

DETAILED DISCLOSURE OF THE INVENTION

As utilized herein, reference to a "neutral-alkaline cellulase" refersto a cellulase composition which retains significant enzymatic activityat pH valves. of about 5.5 and above. In a preferred embodiment, theneutral and/or alkaline cellulase compositions of the subject inventionhave peak enzymatic activity between about pH 5.5 to about 7.5 at 40° C.to about 60° C. In the event that the peak enzymatic activity is at a pHof less than about 5.5, the neutral-alkaline cellulase composition willhave at least about 50% of the optimal enzymatic activity at about pH6.0 to about 7.0 at about 40° C. to about 600° C. By way of example suchactivities may be measured by RBBCMCase, CMCase, endoviscometric orfilter paper activity (FPA). Thus, the cellulase compositions of thesubject invention will have useful enzymatic activity at pHs greaterthan 5.5 such that the enzyme composition can be used in stone wash,detergent, de-inking or other applications where neutral and/or alkalinecellulase activity is needed.

The subject invention relates to compositions of cellulases having highactivity at neutral or alkaline pH's and to unique methods for producingsaid neutral and alkaline cellulase compositions. The neutral/alkalinecellulase compositions of this invention may be obtained from anyspecies of Chrysosporium. In a particularly preferred embodiment, thecellulase compositions of the present invention are isolated fromChrysosporium lucknowense Garg 27K (designated isolate C1) depositedunder the Budapest Treaty with the International Depository at theAll-Russian Collection of Microorganisms of the Russian Academy ofSciences, Bakhrushina St. 8: Moscow, Russia 113184, on Aug. 29, 1996,and assigned accession number VKM F-3500D. The cellulase compositions ofthe subject invention are highly advantageous because they possessenzymatic activity at neutral and/or alkaline pH thereby providingbeneficial performance characteristics in industrial applications.

The cellulase compositions prepared from fungal strains of the subjectinvention exhibit activity at between about pH 5.0 to about 12.0 atbetween about 40° to 60° C. as determined by a CMCase, RBBCMCase orendoviscometric assays. In a preferred embodiment for a stone washprocedure, the cellulase composition may have optimal activity atbetween about pH 5.5 to 7.0 at about 40° C. to about 60° C. Goodperformance activity at neutral and alkaline pH (ie: 6.0, 7.0 & 8.0) hasbeen demonstrated for the neutral and/or alkaline cellulases of theinstant invention in Stonewash application trials and at pH 10.0 andabove for detergent application trials.

The fermentation procedures for culturing cellulolytic microorganismsfor production of cellulase are known in the art. For example, cellulasesystems can be produced either by solid or submerged culture, includingsolid state, batch, fed-batch, and continuous-flow processes. Thecollection and purification of the cellulase systems from thefermentation broth can also be effected by procedures known in the art.The cellulase composition is readily isolated from the fungal cultureby, for example, centrifugation or filtration steps and concentration ofthe filtrate via membrane or hollow fibers ultrafiltration equipment.

The fungal strain Chrysosporium used to produce the cellulasecompositions of the subject invention can be cultured according tostandard methods and conditions known in the art. In a preferredembodiment, the cellulase composition of the subject invention isobtained from the C1 strain. The C1 Chrysosporium strain may be grown ina medium containing inorganic salts, organic nitrogen sources, such aspeptones, defatted cotton seed flour, corn steep liquor, or yeastextract and carbon source. Examples of carbon source include, but is notlimited to, glucose, lactose, sucrose, cellulose or other carbohydrates.More preferably, the fungal strain is grown in media containing bothlactose and peptone or lactose and yeast extract. By way of example thefermentation media can compose lactose at about 0.3% to about 1.0%,preferably about 0.5% to about 0.6%, peptone at about 0.3% to about1.0%, preferably about 0.5% to about 0.6%. Other nitrogen sources andcarbohydrate sources known in the art may be used in the fungal growthmedia including, but not limited to, sweet beet pulp, barley malt, wheatbran, and others known in the art. By way of example sweet beet pulpconcentrate may be used in a range of about 15 to about 30 grams/liter(g/L), preferably about 20 to about 25 g/L; barley malt may be used in arange about 10 g/L to about 20 g/L, preferably about 14 g/L or about 16g/L, wheat bean may be used in a range about 3 g/L to about 8 g/L,preferably about 5 g/L to about 6 g/L. In one embodiment, the C1 strainis cultured in rotated shake flasks in saline medium containing sweetbeet pulp, barley malt, and wheat bran. Cellulase compositions may beisolated from fungi cultured about 3 to 7 days in a growth medium bycentrifugation and ultrafiltration concentration of the cell culturemedium.

Alternatively the Chrysosporium cultures can be cultured on a largescale for commercial use, by using conventional fermentation techniques.In this context fermentation is used broadly to refer to any controlledfungal culturing conditions. Prior to large scale growth an inoculum ofsaid growth culture is generally cultured. The inoculum media maycontain conventional ingredients including, but not limited to, carbonsources, organic nitrogen sources, and inorganic salts. Carbon sourcesmay include, but are not limited to, glucose, lactose, glycerol, and/orcellulose at concentrations in the range of about 0.5 to 200 g/L, morepreferably in the range of about 5 to 50 g/L. Organic nitrogen sourcesmay include, but are not limited to, yeast extract, peptone, or defattedcotton seed flour at concentrations in the range of about 0.5 to 30 g/L,more preferably in the range of 5 to 15 g/L. Inorganic salts mayinclude, but are not limited to, potassium phosphate, for example atabout 0.01 to about 10 g/L, magnesium sulfate, for example at about 0.01to 3.0 g/L, ferrous sulfate, for example at about 0.001 to 10 mg/L.

An inoculum or starter culture may be used to initiate the Chrysosporiumculture for a fermenter by methods known in the art. The media used forfermentation may comprise conventional ingredients for culturing fungi,including but not limited to, cellulose, organic nitrogen sources,magnesium chloride and calcium chloride. Examples of organic nitrogensources include, but are not limited to, peptone or defatted cotton seedflour, such as Pharmamedia.

By way of example, the media may comprise about 5 g/l to about 20 g/L ofpeptone or defatted cotton seed flour, about 10 g/L to about 30 g/L ofcellulose, about 0.03 g/L to about 0.06 g/L of magnesium sulfateheptahydrate and about 0.4 g/L to about 0.8 g/L of calcium chloridedihyrate.

One of skill in the art will appreciate that during fermentation thetemperature, oxygenation, pH, and nutrient levels of fermentationmixture should be maintained. By way of example, dissolved oxygen levelsshould be maintained at about 10 to 60% of air saturation, preferably atabout 20 to 40% of air saturation. The pH should be maintained betweenabout 5 and 8, preferably between about 6.5 and 7.5, most preferablybetween 6.9 and 7.1 and the temperature may be maintained at betweenabout 25° C. to about 40° C., preferably at about 28° C. to 35° C. Thefeed solution may comprise ingredients similar to the fermentation mediabut at higher concentrations to minimize dilution when added to thefermentation media.

The cellulase compositions produced according to the methods of thesubject invention are useful for a variety of other applications forwhich cellulase activity, in particular neutral and/or alkalinecellulase activity, is needed. In one embodiment of this invention, theneutral and/or alkaline cellulase compositions can be used in stonewashing procedures for denim jeans. By way of example, the mostpreferred pH range of stone wash applications is between about 5.5 to7.5, most preferably at about pH 6 to about 7. The neutral and/oralkaline cellulase composition obtained from Chrysosporium isolatesadvantageously have significant enzymatic activity at or above neutralor alkaline pH. Stone wash procedures conducted with neutral and/oralkaline cellulase run at neutral and/or alkaline pH's are particularlyadvantageous compared to traditional procedures using acid cellulases(eg: those from Trichoderma reesei) because of lower levels ofbackstaining on the garments, less strength loss to the garments and thealkalinity of the water that is present naturally during this process.These stone washing procedures result in jeans with highly desirablefeel and appearance. By way of example, 0.02 to 10 g of cellulasepreparation 47.0528 described herein, may be used per 135 g of denim.One of skill in the art will know how to regulate the amount orconcentration of the cellulase composition produced by this inventionbased on such factors as the activity of the cellulase, and the washconditions, including but not limited to temperature and pH.

In yet another embodiment of this invention, the cellulase compositionsof this invention can be used to reduce or eliminate the harshnessassociated with fabrics made from cellulose by addition to detergentcompositions. By way of example, the preferred range for detergentcompositions is between about pH 8 to about 12, most preferably pH 10 toabout 11. The cellulase compositions of the subject invention can beused in detergent compositions at neutral and or alkaline pH. Detergentingredients contemplated for use with the cellulase composition of thesubject invention include any detergent ingredient known in the art.Examples of such ingredients include, but are not limited to,detergents, buffers, surfactants, bleaching agents, softeners, solvents,solid forming agents, abrasives, alkalis, inorganic electrolytes,cellulase activators, antioxidants, builders, silicates, preservatives,and stabilizers, and are known in the art. The detergent compositions ofthis invention preferably employ a surface active agent, i.e.,surfactant, including anionic, non-ionic, and ampholytic surfactantswell known for their use in detergent compositions. In addition to thecellulase components and the surface active agent, the detergentcompositions of this invention can additionally contain one or more ofthe following components; the enzymes amylases, cellulases, proteinase,and lipases; cationic surfactants and long-chain fatty acids; builders;antiredeposition agents; bleaching agents; bluing agents and fluorescentdyes; caking inhibitors; masking agents for factors inhibiting thecellulase activity; cellulase activators; antioxidants; andsolubilizers. In addition, perfumes, preservatives, dyes, and the likecan be used, if desired, with the detergent compositions of thisinvention. Examples of detergent compositions employing cellulases areexemplified in U.S. Pat. Nos. 4,435,307; 4,443,355; 4,661,289;4,479,881; 5,120,463, which are herein incorporated by reference.

When a detergent base used in the present invention is in the form of apowder, it may be one which is prepared by any known preparation methodincluding a spray-drying method and/or a granulation method. Thegranulation methods are the most preferred because of the non-dustingnature of granules compared to spray dry products. The detergent baseobtained by the spray-drying method is hollow granules which areobtained by spraying an aqueous slurry of heat-resistant ingredients,such as surface active agents and builders, into a hot space. Thegranules have a size of from about 50 to about 2000 micrometers. Afterthe spray-drying, perfumes, enzymes, bleaching agents, and/or inorganicalkaline builders may be added. With a highly dense, granular detergentbase obtained by such as the spray-drying-granulation method, variousingredients may also be added after the preparation of the base. Whenthe detergent base is a liquid, it may be either a homogenous solutionor an inhomogeneous solution.

The cellulase compositions of this invention preferably exhibit highlevels of activity at alkaline or neutral pH's, but also may exhibitenzymatic activity at acidic pH's. Therefore, the detergent compositionscomprising the cellulases of the present invention can be used in abroad pH range of from acidic to alkaline pH.

Other textile applications in which these cellulase compositions may beused include, but are not limited to, Garment Dyeing applicationsincluding but not limited to Enzymatic Mercerizing of viscose,Bio-Polishing applications, Enzymatic Surface Polishing; Biowash(washing or washing down treatment of textile materials), EnzymaticMicrofibrillation, Enzymatic "cottonization" of linen, ramie and hemp;and treatment of Lyocel or Newcell (ie; "TENCEL" from Courtauld's),Cupro and other cellulosic fibers or garments, dye removal from dyedcellulosic substrates such as dyed cotton (Leisola & Linko--(1976)Analytical Biochemistry, v. 70, p. 592. Determination Of TheSolubilizing Activity Of A Cellulase Complex With Dyed Substrates; Blum& Stahl--Enzymic Degradation Of Cellulose Fibers; Reports of theShizuoka Prefectural Hamamatsu Textile Industrial Research Institute No.24 (1985)), as a bleaching agent to make new indigo dyed denim look old(Fujikawa--Japanese Patent Application Kokai No. 50-132269), to enhancethe bleaching action of bleaching agents (Suzuki--Great Britain PatentNo. 2 094 826), and in a process for compositions for enzymatic desizingand bleaching of textiles (Windbichtler et al., U.S. Pat. No. 2,974,001.Another example of enzymatic desizing using cellulases is provided inBhatawadekar (May 1983) Journal of the Textile Association, pages 83-86.

In other industrial embodiments, the cellulase compositions can be usedin the saccharification of lignocellulose biomass from agriculture,forest products, municipal solid waste, and other sources, for theproduction of fuels and other chemicals through fermentation, forbiobleaching of wood pulp, and for de-inking of recycled print paper allby methods known to one skilled in the art.

In yet another embodiment of the subject invention, various componentsof the neutral and alkaline cellulase compositions can be isolated andused independently of each other. Specific components or cellulasecomposition enriched by certain cellulase components can be produced orisolated by chemical and physical means from mutants or specificallyproduced by genetic engineering methods. The cellulase system can bepurified into separate components by art-recognized separationtechniques including ion exchange chromatography at a suitable pH,affinity chromatography, size exclusion, chromatography and like. Forexample, in ion exchange chromatography, it is possible to separate thecellulase components by eluting with a pH gradient, or a salt gradient,or both. Such separations can be done by those skilled in the art havingthe benefit of the teachings provided herein.

Once the individual enzymatic components of the cellulase compositionare fractionalized and isolated the proteins may be partially sequencedor microsequenced to design synthetic DNA or probes to isolate the geneencoding the enzymatic proteins of interest. Generally the aminoterminal sequence of the protein is determined by conventional proteinsequencing methods or by automated sequence (Ausubel et al., (1987) in"Current Protocols in Molecular Biology", John Wiley and Sons, New York,N. Y.). Alternatively, other regions of the protein may be sequenced incombination with chemical cleavage or enzymatic cleavage and proteinseparation techniques. (Ausubel et al., (1987) in "Current Protocols inMolecular Biology", John Wiley and Sons, New York, N.Y.). One of skillin the art will understand that the synthetic DNA clones or probes canbe used in routine cloning techniques to isolate the genes correspondingto the enzymes present in the neutral/alkaline cellulase compositionsproduced by Chrysosporium.

It will be understood by one skilled in the art that nucleic acidsequences obtained by this invention in the art may vary due to thedegeneracy of the genetic code variations in the DNA sequence, but willstill result in a DNA sequence capable of encoding the enzymaticcomponents of the cellulase compositions. Such DNA sequences aretherefore functionally equivalent to the nucleic acid sequences of theinstant invention and are intended to be encompassed within the presentinvention. Also intended to be encompassed within this invention arenucleic acid sequences which are complementary to nucleic acid sequencescapable of hybridizing to the disclosed nucleic acid sequence under avariety of conditions.

This invention further includes nucleic acid sequences encoding theenzymes of the cellulase compositions of this invention and thoseproteins or peptides having substantially the same function as theenzymatic proteins or peptides of this invention. Such proteins orpolypeptides include, but are not limited to, a fragment of the protein,or a substitution, addition or deletion mutant. This invention alsoencompasses proteins or peptides that are substantially homologous tothe proteins encoding the enzymes comprising the cellulase compositionof this invention. The term "analog" includes any polypeptide having anamino acid residue sequence substantially identical to the sequencespecifically in which one or more residues have been conservativelysubstituted with a functionally similar residue and which displays thefunctional aspects of the proteins as described herein. Examples ofconservative substitutions include the substitution of one non-polar(hydrophobic) residue such as isoleucine, valine, leucine or alanine foranother, the substitution of one polar (hydrophilic) residue for anothersuch as between arginine and lysine, between glutamine and asparagine,between threonine and serine, the substitution of one basic residue suchas lysine, arginine or histidine for another, or the substitution of oneacidic residue, such as aspartic acid or glutamic acid for another.

Proteins or polypeptides of the present invention also include anypolypeptide having one or more additions and/or deletions or residuesrelative to the sequence of a polypeptide whose sequence is included inthe proteins of this invention so long as the requisite activity ismaintained.

This invention also provides a recombinant DNA molecule comprising allor part of the nucleic acid sequences isolated by this invention and avector. Expression vectors suitable for use in the present inventioncomprise at least one expression control element operationally linked tothe nucleic acid sequence. The expression control elements are insertedin the vector to control and regulate the expression of the nucleic acidsequence. Examples of expression control elements include, but are notlimited to, lac system, operator and promoter regions of phage lambda,yeast or other fungi promoters. Examples of promoters that may be usedinclude, but are not limited to, glucoamylase. Additional preferred orrequired operational elements include, but are not limited to, leadersequence, termination codons, polyadenylation signals and any othersequences necessary or preferred for the appropriate transcription andsubsequent translation of the nucleic acid sequence in the host system.It will be understood by one skilled in the art the correct combinationof required or preferred expression control elements will depend on thehost system chosen. It will further be understood that the expressionvector should contain additional elements necessary for the transfer andsubsequent replication of the expression vector containing the nucleicacid sequence in the host system. Examples of such elements include, butare not limited to, origins of replication and selectable markers. Itwill further be understood by one skilled in the art that such vectorsare constructed using conventional methods (Ausubel et al., (1987) in"Current Protocols in Molecular Biology", John Wiley and Sons, New York,N.Y.) or may be commercially available.

Another aspect of this invention relates to a host organism into whichrecombinant expression vector containing all or part of the nucleic acidsequence has been inserted. The host cells transformed with the nucleicacid sequence of this invention includes eukaryotes, such as animal,plants or seeds, insect and yeast cells, fungal cells, and prokaryotes,such as E. coli or other bacteria. Examples of fungal host cells includebut are not limited to Aspergillus, Trichoderma, Humicola, Pencillium,or Neurospora. The means by which the vector carrying the gene may beintroduced into the cell include, but are not limited to,transformation, microinjection, electroporation, transduction, ortransfection using DEAE-dextran, lipofection, calcium phosphate or otherprocedures known to one skilled in the art (Sambrook et al. (1989) in"Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press,Plainview, N.Y.). Alternatively, Chrysosporium cells can be transformedwith the nucleic acid sequence of this invention to amplify productionof cellulases by Chrysosporium.

In a preferred embodiment, expression vectors that function in fungalcells are used. Examples of such vectors include, but are not limited toplasmids, described in the patents (Ogawa; Japanese patent JP5095787 A930420, Ozeki; Japanese patent JP7115976 A 950509, Murakami; Japanesepatent JP3094690 A 910419, Ishida; Japanese patent JP3251175 A 911108,Uozumi; Japanese patent JP5268953 A 931019 DW9346 C12N-009/34011pp,Gottschalk; German patent DE3908813 A 900920 DW9039 000 pp, Gysler;European patent EP-683228 A2 951122 DW9551 C12n-015/60 Eng 041 pp). Itis preferred that the recombinant protein expression vector isintroduced into fungal cells, such to ensure proper processing andmodification and modification of the introduced protein.

In a further embodiment, the recombinant protein expressed by the hostcells can be obtained as a crude lysate or can be purified by standardprotein purification procedures known in the art which may includedifferential precipitation, molecular sieve chromatography, ion-exchangechromatography, isoelectric focusing, gel electrophoresis, affinity, andimmunoaffinity chromatography and the like. (Ausubel et. al., (1987) in"Current Protocols in Molecular Biology" John Wiley and Sons, New York,N.Y.). In the case of immunoaffinity chromatography, the recombinantprotein may be purified by passage through a column containing a resinwhich has bound thereto antibodies specific for the protein of interest(Ausubel et. al., (1987) in "Current Protocols in

Molecular Biology" John Wiley and Sons, New York, N.Y.).

All or parts of the nucleic acid sequences of this invention can also beused as probes to isolate other homologs in other genera or strains. Ina preferred embodiment the nucleic acid sequences are used to screen aChrysosporium library; positive clones are selected and sequenced.Examples of sources from which the gene library can be synthesizedinclude, but are not limited to species of Chrysosporium, Aspergillus,Pencillium, Humicola, Cephalosporium Tricoderma or bacteria such asBacillus. One skilled in the art will understand the appropriatehybridization conditions to be used to detect the homologs. Conventionalmethods for nucleic acid hybridization, construction of libraries andcloning techniques are described in Sambrook et al., (eds) (1989) In"Molecular Cloning A Laboratory Manual" Cold Spring Harbor Press,Plainview, N.Y. and Ausubel et al., (eds) in "Current Protocols inMolecular Biology" (1987), John Wiley and Sons, New York, N.Y.

This invention also relates to mutant strains of Chrysosporium, inparticular, mutant strains of Chrysosporium lucknowense capable ofproducing neutral and/or alkaline cellulases. Methods of DNA mutagenesisand screening for mutants are well known to these skilled in the art andinclude a variety of chemical and enzymatic methods. Examples of suchmethods include but are not limited to exposure of the fungi toultraviolet light (UV), nitrous acid,N-methyl-N'-nitro-N-nitrosoguanidine (NG), and 4-nitroquinoline-N-oxide(4NQO). (Leninger (1972) Biochemistry, Worth Publishers Inc., N.Y.;Jeffrey H. Miller (1972) "Experiments in Molecular Genetics. Cold SpringHarbor Laboratory, Cold Springs Harbor, N.Y.). Preferred methods,include UV and NG. By way of example, mutant strains of Chrysosporiumcapable of producing increased levels cellulase compositions exhibitingenzymatic activity at neutral and or alkaline pH's may be generated byUV mutagenesis. By way of examples mutants may be produced by exposureof the fungal spores to UV light for a period of about 10 to about 180seconds, preferably 45 to about 90 seconds, and most preferably 65 to 75seconds. Mutageneses involving NG may involve varying the concentrationof the NG and exposure time. By way of example NG, at about 13milligrams/liter (mg/L) to about 400 mg/L of NG may be used for a periodof exposure of about 15 minutes to about 120 minutes. Alternativemethods for generating mutants include techniques in the field ofmolecular biology. Examples of such techniques include, but are notlimited to, oligonucleotide directed mutagenesis, linker scanningmutations or oligonucleotide directed mutagenesis using polymerase chainreaction (Ausubel (1987) Current Protocols in Molecular Biology).

Screening and selection of mutant Chrysosporium clones exhibitingimproved cellulase production can also be performed by conventionalmethodology. Examples of selection criteria for screening mutantsincludes, but is not limited, to the ability of the fungal colony tobreakdown cellulase as evidenced by cellulose clearing by the fungigrown on media plates containing cellulose and increased growth rate onmedia containing cellulose. By way of example, after mutagenesis thefungal sample may be plated on agar plates containing cellulose byconventional methodology. Colonies exhibiting increased growth andcellulose clearing (as is evidenced by clearing of the media surroundingthe isolate) may be selected. However, any other assay for celluloseclearing may be used. Once a mutant is isolated the stock or sporesderived from the mutant may be maintained by conventional methodology.The mutants isolated by this method may be cultured or fermented underthe conditions described herein and above for the wild type strains ofChrysosporium. The individual enzymatic components of the cellulasecomposition produced by the mutants may be partially sequenced ormicrosequenced to design synthetic to isolate the genes encoding theenzymatic proteins of interest. Thus, this invention relates to thenucleic acid sequences encoding the cellulase enzymes produced by theChrysosporium mutants and to the enzymes themselves. In yet anotherembodiment of this invention, the mutants isolated may be subjected tofurther mutagenisis and screened by the methods described herein above.

All books, patents or articles references herein are incorporated byreference. The following examples illustrate various aspects of theinvention but in no way are included to limit the invention. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Materials and Methods

Enzyme assays. The assay for CMCase, used carboxymethyl cellulose as theenzymatic substrate, measured the initial rate of hydrolysis, andquantified the amount of reducing sugars released according to themethod of Somogyi and Nelson. The method is described in Methods inEnzymology, Vol. 160A, pp. 102-104. Endo-1,4-β-glucanase activity wasassayed viscometrically as a rate of the decrease of viscosity ofsoluble substrate CMC according to Bioorganicheskaya Khimia, Vol. 6, pp.1225-1242 (endoviscometric assay). Filter paper activity (FPA) or totalcellulase activity used filter paper as the substrate and estimated theactivity required to release 2 mg glucose from a 50-mg sample of filterpaper. The assay is based on the Commission on Biotechnology (IUPAC) forthe measurement for total cellulase activity or true cellulase and isdescribed in Methods in Enzymology, Vol. 160A, pp. 94-97. Avicelaseactivity was estimated as initial rate of reducing sugar formationduring hydrolysis if Avicell-cellulose (as described in BioresourceTechnology, Vol. 52, pp. 119-124). The assay of cellobiase usedcellobiose as the substrate and measured the amount of glucose released(described in Methods in Enzymology, Vol. 160A, pp. 111-112). The assayof β-glucosidase activity used p-nitrophenyl-β-D-glucoside as thesubstrate (described in Methods in Enzymology, Vol. 160A, pp. 109-1100).The protein was determined by Lowry method (according to Journal ofBiological Chemistry, Vol. 193, pp. 165-175). The assay of RBB-CMCase isbased on determination of the dye release from soluble substrate RBB-CMC(CMC, dyed with Remazolbrilliant Blue), reference to assay--see Megazyme(Australia) Ltd., Product Information Bulletin, April, 1995.

EXAMPLE 1 Isolation of C1 Strain

The strain was isolated from samples of forest alkaline soil from SolaLake, Far East of Russian Federation (Pacific Coast of Russia, about5000 miles east from Moscow). A mixed soil sample was collected from 10different sites. One gram of each sample was transferred into a flaskwith 100 ml sterile tap water and sonicated with an ultrasonic dispenserfor 1 minute (0.44 Amp, 22 KHz). The suspension (diluted 1:500) wasinoculated into petri dishes with Czapek medium (pH 5.5-6.0) containing100 mg/L streptomycin. The study was conducted in three replicates.Colonies of various color shape and size were identified for a secondisolation step. Further isolation of the sample was performed on plateswith Czapek media, malt agar, potato dextrose agar, or Getchinson salinemedium pH 7.5 (Table 2). Plates were incubated at about 28° C. forseveral days. Selection for cellulase producers was performed oncellulose agar plates which contained the components shown in Table 1.Preparation of amorphous cellulose is described in Methods in Enzymologyvol. 160A.

                  TABLE 1                                                         ______________________________________                                        Cellulose agar plates                                                         Ingredients       g/L                                                         ______________________________________                                        KH.sub.2 PO.sub.4 1                                                           KCl               0.1                                                         MgSO.sub.4.7H.sub.2 O                                                                           0.3                                                         NaCl              0.1                                                         FeCl.sub.3        0.01                                                        NaNO.sub.3        2.5                                                         Amorphous cellulose                                                                             5                                                           Agar              15                                                          pH                7.5                                                         ______________________________________                                    

The plates were incubated for 3-7 days at 28° C. The formation of lightclearing halos around the colonies indicated cellulase activity. Onestrain, designated herein as C1, that exhibited significant levels ofcellulase activity was chosen for additional study. The strain wasdeposited at the All-Russian Collection of Microorganisms of RussianAcademy of Sciences, (VKM), abbreviation in English - RCM), BakhrushinaSt. 8: Moscow, Russia, 113184 under the Budapest Treat on Aug. 29, 1996,as Chrysosporium lucknowense Garg 27K, VKM-F 3500 D).

EXAMPLE 2 Characterization of C1 Strain

Growth of the C1 strain on potato dextrose agar gives colonies of 55-60mm diameter after 7 days. C1 colonies exhibit a white-cream color, thesurface is velvet-like and has a slightly raised center. The edge of thecolonies is a flat, thin and fibereil. The back side of the colonies hasa light cream color.

The mycelium has hyaline and is slightly branched and smooth. The hyphaeare thin-walled. Air hyphae are septate and form spores of 2.0-3.0micrometers width; the substrate hyphae are sterile.

The conidia are terminal and lateral. No intercalary conidia were found.The majority of conidia are connected with hyphae through short stems orshort side branches. The conidia are separated but adjacent. Conidia arehyaline, thin-walled, oval or clavate, and single cellular. Their sizevaries from 4 to 10 micrometers in diameter.

The C1 strain can be maintained on malt extract agar (at 4° C.), andtransferred each six months. Maintenance in liquid nitrogen and bylyophilization is also possible. The C1 strain is haploid, filamentous,can grow on agar plates with growth restricting agents like bovinebile(1.5%), and produces spores.

EXAMPLE 3 Classification of C1 Strain

According to Sutton classification (Van Dorschot, C.A.N. 1980! "Arevision of Chrysosporium and allied genera," in Studies in Mycology,No. 20, Centraaddbureau voor Schimmelcultures, Baarn, The Netherlands,pp. 1-36), the C1 strain of the subject invention belongs to the orderof Hyphomycetales, family of Moniliaceae, genus of Chrysosporium,species of Chrysosporium lucknowense Garg 1966. This classification wasbased on observation of the following characteristics of the C1 strain:

1. Signs of Hyphomycetales order. Conidia are produced directly onmycelium, on separate sporogenous cells or on distinct conidiophores.

2. Signs of Moniliaceae family. Both conidia and conidiophores (ifpresent) are hyaline or brightly colored; conidiophores are single or inloose clusters.

3. Signs of Chrysosporium Corda 1833 genus. Colonies are usuallyspreading, white, sometimes cream-colored, pale brown or yellow, feltyand/or powdery. Hyphae are mostly hyaline and smooth-walled, withirregular, more or less orthotopic branching. Fertile hyphae exhibitlittle or no differentiation. Conidia are terminal and lateral, thallic,borne all over the hyphae, sessile or on short protrusions or sidebranches, subhyaline or pale yellow, thin- or thick-walled, subglobose,clavate, pyriform, orobovoid, 1-celled, rarely 2-celled, truncate.Intercalary conidia are sometimes present, are solitary, occasionallycatenate, subhyaline or pale yellow, broader than the supporting hyphae,normally 1-celled, truncate at both ends. Chlamydospores areoccasionally present.

4. Signs of Chrysosporium lucknowense Garb 1966 species. Colonies attain55 mm diameter on Sabouraud glucose agar in 14 days, are cream-colored,felty and fluffy; dense and 3-5 mm high; margins are defined, regular,and fimbriate; reverse pale yellow to cream- colored. Hyphae arehyaline, smooth- and thin-walled, little branched. Aerial hyphae aremostly fertile and closely septate, about 1-3.5 mm wide. Submergedhyphae are infertile, about 1-4.5 mm wide, with the thinner hyphae oftenbeing contorted. Conidia are terminal and lateral, mostly sessile or onshort, frequently conical protrusions or short side branches. Conidiaare solitary but in close proximity to one another, 1-4 conidiadeveloping on one hyphal cell, subhyaline, fairly thin- andsmooth-walled, mostly subglobose, also clavate orobovoid, 1-celled,2.5-11×1.5-6 mm, with broad basal scars (1-2 mm). Intercalary conidiaare absent. Chlamydospores are absent.

5. Description of C1 strain. Colonies grow to about 55-60 mm diameter insize on potato-dextrose agar in about 7 days; are white-cream-colored,felty, 2-3 mm high at the center; margins are defined, regular,fimbriate; reverse pale, cream-colored. Hyphae are hyaline, smooth- andthin-walled, little branched. Aerial hyphae are fertile, septate, 2-3 mmwide. Submerged hyphae are infertile. Conidia are terminal and lateral;sessile or on short side branches; absent; solitary, but in closeproximity to one another, hyaline, thin- and smooth-walled, subglobose,clavate or obovoid, 1-celled, 4-10 mm. Chlamydospores are absent.Intercalary conidia are absent.

Conclusion. C1 is a strain of Chrysosporium lucknowense Garg 1966. Forconvenience the cellulase made by this strain is referred to herein as"C1" or "C1 cellulase."

EXAMPLE 4 Assay for Cellulase Activity

The C1 strain was grown in 800 ml shake flasks rotated at 220 rpm andincubated at 28° C. The C1 strain was grown in saline Getchinson medium(See Table 2) (pH 7.5) containing 5 g/L of various nutrients, and insome cases with 2 g/L microcrystalline cellulose. One hundred ml ofmedia were added to each flask.

                  TABLE 2                                                         ______________________________________                                        Getchinson medium for shake flasks                                                          g/L                                                             ______________________________________                                               KH.sub.2 PO.sub.4                                                                      1                                                                    KCl      0.1                                                                  MgSO.sub.4.7H.sub.2 O                                                                  0.3                                                                  NaCl     0.1                                                                  FeCl.sub.3                                                                             0.01                                                                 NaNO.sub.3                                                                             2.5                                                           ______________________________________                                    

Combinations of glucose and microcrystalline cellulose, dextrose andmicrocrystalline cellulose, glycerol and microcrystalline cellulose,lactose and microcrystalline cellulose resulted in very low growth,formation of large aggregates of mycelium, and in the absence ofcellulase activities (CMC-ase assays). The results are presented inTable 3. Additions of nitrogen organic sources, i.e., peptone, cornsteep liquor, or yeast extract enhanced growth and cellulase productionand did not result in mycelium aggregates.

Lactose and yeast extract gave the highest cellulase production by C1.Similar results were obtained when the lactose and yeast extract weresubstituted with 25 g/L sweet beet pulp, 15 g/L barley malt, and 5 g/Lwheat bran.

                  TABLE 3                                                         ______________________________________                                        Effect of carbon and nitrogen sources on CMC-ase activity                     of C1 (shake flasks results)                                                                CMC-ase activity (units/ml) at pH 7.0                                         Days                                                            Substrate       3        5          7                                         ______________________________________                                        Glucose + cellulose                                                                           0        0          0                                         Dextrose + cellulose                                                                          0        0          0                                         Glycerol + cellulose                                                                          0        0          0                                         Lactose + cellulose                                                                           0        0          0                                         Lactose + corn steep liquor                                                                   0        0          0.9                                       Lactose + peptone                                                                             10.7     7.4        14.8                                      Lactose + yeast extract                                                                       0        18.5       10.0                                      Cellulose + peptone                                                                           0.3      1.2        1.6                                       Cellulose + corn steep liquor                                                                 1.9      2.8        5.5                                       ______________________________________                                    

EXAMPLE 5 Production of Cellulase for Stone Wash Tests

1. Production in shake flasks. C1 strain was grown in 800 ml shakeflasks rotated at 220 rpm and incubated at 28° C. for seven days. Thegrowth medium 100 ml per flask was saline Getchinson medium (see Table2) (pH 7.5) containing 25 g/L sweet beet pulp, 15 g/L barley malt, and 5g/L wheat bran. The cell mass was separated by centrifugation and thecell-free supernatant was lyophilized and stored for further tests. C1cellulase preparation #s 47.1.1 to 47.15.1 were produced in this manner.C1 preparation #47.16.1 was produced by the same manner, but cell-freesupernatant after centrifugation was ultrafiltrated using a 10 kDacutoff membrane before lyophilization. C1 preparation #'s 47.18.1 to47.22.1 were produced by the same manner in shake flasks with Getchinsonmedium, but containing lactose (0.5% w/v) and peptone (0.5% w/v) insteadof sweet beet pulp, barley malt and wheat bran. The cell mass wasseparated by centrifugation and the cell free supernatant waslyophilized and stored for further tests. Preparation #'s 47.1000,47.1001, 47.2000 & 47.2001 were produced in shake flasks by the samemanner as preparation #'s 47.1.1-47.15.1 except that they were producedusing other Chrysosporium strains. Specifically, 47.2001 was produced byChrysosporium pannorum, preparation 47.2000 was produced byChrysosporium pruinosum, preparation 47.1001 was produced byChrysosporium keratinophilim and preparation 47.1000 was produced byChrysosporium queenslandicum (see Example 8). The protein content andactivity fingerprints of these C1 preparations are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        Protein content and activity fingerprints of C1 preparations and              preparation #'s 47.1000, 47.1001, 47.2000 & 47.2001 which were                prepared from other species of Chrysosporium sp.                                                    CMC-   Endo         β-Glu                          Prepara-                                                                            Protein,                                                                              FPA,    ase,   (visc),                                                                             Avice- cosidase,                           tion #                                                                              %       FPU/g   U/g    U/g   lase, U/g                                                                            U/g                                 ______________________________________                                        47.1.1                                                                              22      13      170    120   23     135                                 47.2.1                                                                              26      14      137    110   22     190                                 47.3.1                                                                              15      19      140    128   18     198                                 47.4.1                                                                              18      23      150    133   55     220                                 47.5.1                                                                              16      20      179    120   71     185                                 47.6.1                                                                              17      22      224    134   82     280                                 47.7.1                                                                              8       4       78     123   10     22                                  47.8.1                                                                              22      14      168    123   19     124                                 47.9.1                                                                              28      15      204    174   23     151                                 47.10.1                                                                             24      11      181    185   16     147                                 47.11.1                                                                             28      16      234    191   25     269                                 47.12.1                                                                             26      14      167    138   20     178                                 47.13.1                                                                             25      9       137    110   13     141                                 47.14.1                                                                             15      6       39     33    9      59                                  47.15.1                                                                             14      6       95     44    10     75                                  47.16.1                                                                             16      10      146    39    15     107                                 47.17.1                                                                             7       3       100    34    5      29                                  47.18.1                                                                             10      30      120    38    10     42                                  47.19.1                                                                             14      4       28     10    4      11                                  47.20.1                                                                             14      6       17     5     1      9                                   47.21.1                                                                             13      3       34     5     3      9                                   47.22.1                                                                             14      5       35     6     3      10                                  47.1000                                                                             18      4       31     35    6      89                                  47.1001                                                                             13      6       103    38    10     66                                  47.2000                                                                             10      3       78     31    7      67                                  47.2001                                                                             13      3       45     39    7      4                                   47.0325                                                                             50      155     4965   964   184    248                                 47.0528                                                                             67      111     13500  1782  232    423                                 ______________________________________                                    

2. Production in fermentors. C1 cellulase was produced in a 10-L"ANKUM-1M" fermentor with Getchinson medium, lactose (0.5% w/v), peptone(0.5% w/v), and chloramphenicol (50 mg/mL). Initial volume of thenutrition medium was 7.0 L, final volume after fermentation was 7.3 L.The dissolved oxygen concentration (DO), agitation speed, aerationlevel, temperature, and pH were controlled. Fermentation was carried outas a batch-mode. The temperature of the fermentation was controlled at28° C. The initial pH was 7.5 and was later maintained at that level byaddition of NH₄ OH (12% w/v). The aeration was at 4-5 L/minute andagitation at 400-500 rpm. The DO was maintained at above 50% Samples (30ml) were taken for analysis every 8 hours. At the end of fermentation,fungal biomass was separated by centrifugation (10,000 g, roomtemperature, 20 minutes), and culture filtrate was lyophilized andstored for further tests. The results are shown in Table 5. Celluasepreparation #47.17.1 was produced in this manner. Protein content andactivity fingerprint of this C1preparation is shown in Table 4.

                  TABLE 5                                                         ______________________________________                                        Production of C1 cellulase in 10-L fermentor                                  Time (h)                                                                             DO (%)    Reducing sugars (g/L)                                                                       CMC-ase (U/mL)                                 ______________________________________                                         0     100       4.8           0                                               8     90        4,7           0                                              16     54        4.4           0                                              24     66        1.2           4                                              32     70        0.4           10                                             40     73        0.3           11.5                                           48     70        0.1           5                                              56     70        0             1                                              ______________________________________                                    

3. Production of C1 Preparation #'s 47.0325 and 47.0528. C1 cellulasepreparation #47.0325 was produced using the wild type C1 strain,preparation #47.0528 was produced using an improved mutant obtained fromthe wild type C1 strain. These preparations were grown up fermentorsunder the conditions described in Examples 13 and 15. Preparation47.0325 was produced using a batch fermentation and 47.0528 was producedusing a fed batch fermentation protocol.

4. Preparation of Humicola wild type preparation #'s 14.22.1 & 14.23.1The wild type Humicola grisea var. thermoidea preparation #14.22.1 wasproduced from the ATCC 16453 strain and the wild type Humicola insolenspreparation #14.23.1 was produced from the ATCC 16454 strain. TheseHumicola wild type preparations were produced in shake flasks using thesame method as described above for (Production in shake flasks) of C1preparation #'s 47.1.1-190 47.15.1.

EXAMPLE 6 Comparison of C1 to Other Neutral Cellulases

The FPA, CMCase and endoglucanase activities of C1 enzyme preparation#47.0528 were compared to commercial Humicola insolens (Denimax XT) andto wild ATCC-type Humicola (preparation #'s 14.22.1 Humicola grisea var.thermoidea (ATCC 16453) & 14.23.1 Humicola insolen (ATCC 16454) neutralcellulases. The results are given in the Table 6. The total activitiesof C-1 #47.0528 are clearly higher than those of neutral cellulases fromwild type Humicola and from commercial Humicola insolen preparation. Thespecific CMCase and endoglucanase activities (as units per gram of drypreparation or units per gram of protein) of C-1 47.0528 are higher thanthose of all tested Humicola preparations listed in Table 6. Thespecific FPA of C-1 #47.0528 is higher than the specific FPA of Humicolawild type preparations #14.22.1 & 14.23 and slightly lower than thespecific FPA of the Humicola insolen commercial product Denimax XT. ThepH and thermal stability of C1 cellulase was similar to Denimax XT.

                                      TABLE 6                                     __________________________________________________________________________    Comparison of C1 and Humicola cellulases.                                                            Endo       Endo                                                       FPA                                                                              CMCase                                                                             (visc)                                                                           FPA                                                                              CMCase                                                                             (visc)                                                 Protein                                                                           unit/1 gram of dry                                                                       units/1 gram of                                                %   preparation                                                                              protein                                             __________________________________________________________________________    C1 (47.0528)                                                                             67  111                                                                              13,500                                                                             1782                                                                             165                                                                              20,115                                                                             2,655                                       Humicola sp. (#14.23.1)                                                                  10  2  28   30 20 280  300                                         Humicola sp. (#14.23.1)                                                                  10  1  11   19 10 110  190                                         Denimax XT 13  25 450  99 192                                                                              3,460                                                                              761                                         (commercial)                                                                  __________________________________________________________________________     (*) Activities were measured at pH 5.0 and 50° C.                 

EXAMPLE 7 The effect of pH and temperature on activity and stability ofC1 FPA and CMC-ase activities

The FPA and CMC-ase activities of C1 exhibit optimal stability andactivity at about pH 6-7 and about 50°-60° C.; the pH optimum forCMC-ase activity is about 6.5, and the optimum temperature is about 55°C. (see Tables 8, 9). At pH 8.0 (50° C.), CMC-ase possesses 80%activity, and FPA--78% activity, at pH 9.0 (50° C.), CMC-ase possesses65% activity, and FPA--52% activity (see Table 7.).

                  TABLE 7                                                         ______________________________________                                        the effect of pH on FPA and CMCase activities of C1 cellulase                 (#47.19.1) at 50° C.                                                   pH (50° C.)                                                                           FPA (%)  CMC-ase (%)                                           ______________________________________                                        4.0            50       60                                                    4.5            68       70                                                    5.0            75       78                                                    5.5            80       80                                                    6.0            92       90                                                    6.5            100      100                                                   7.0            95       95                                                    7.5            90       92                                                    8.0            78       80                                                    8.5            60       75                                                    9.0            52       65                                                    ______________________________________                                    

The incubation time for the FPA assay was 60 minutes, the incubationtime for CMCase assay was 5 minutes.

                  TABLE 8                                                         ______________________________________                                        The effect of temperature on FPA and CMC-ase activities of C1                 cellulase (#47.19.1), at pH 7.0                                               Temperature (C.)                                                                             FPA (%)  CMC-ase (%)                                           ______________________________________                                        40             45       50                                                    45             60       55                                                    50             70       65                                                    55             100      100                                                   60             70       60                                                    65             40       30                                                    70             20       25                                                    ______________________________________                                    

The incubation time for the FPA assay was 60 minutes, the incubationtime for the CMCase assay was 5 minutes.

                  TABLE 9                                                         ______________________________________                                        Stability of CMC-ase of C1 cellulase (#47.19.1) at 50° C.                      CMC-ase activity remained (%)                                         Time (h)  pH 5.1  pH 7.2      pH 7.7                                                                              pH 8.5                                    ______________________________________                                        0         100     100         100   100                                       0.5       100     98          95    85                                        1         100     95          93    55                                        2         100     82          78    32                                        3         100     78          65    25                                        5         100     75          45    15                                        ______________________________________                                    

The CMC-ase of C1 exhibits high stability at optimal pH and temperature:

For Example; at pH 7.2 and 50° C. CMCase possesses 95% activity after 1hour and 75% activity after 5 hours, at pH 7.7 and 50° C. CMCasepossesses 93% activity after 1 hour and 45% activity after 5 hours (SeeTable 9.).

EXAMPLE 8 Neutral and/or alkaline Cellulase activity/performancedemonstrated in Other Strains of the Same Genera of Chrysosporium

Various strains of the Chrysosporium genus were tested for cellulaseproduction. The full names and origins of these strains are describedbelow.

Strains obtained from the American Type Culture Collection (ATCC),Rockville, Md. include:

1. ATCC 44006 Chrysosporium lucknowense

2. ATCC 34151 Chrysosporium pannorum

3. ATCC 24782 Chrysosporium pruinosum

Strains obtained from the Russian Collection of Microorganisms (VKM)include:

1. VKMF-2119 Chrysosporium keratinophilum

2. VKMF-2875 Chrysosporium keratinophilum

3. VKMF-2120 Chrysosporium lobatum

4. VKMF-2121 Chrysosporium merdarium

5. VKMF-2116 Chrysosporium queenslandicum

6. VKMF-2117 Chrysosporium queenslandicum

7. VKMF-2877 Chrysosporium tropicum

Two types of growth media were used in this study: medium A-GetchinsonGetchinson with sugar beet press, barley malt, and wheat bran; andmedium B-Getchinson with peptone and lactose. The compositions of themedia are described in Table 11.

                  TABLE 11                                                        ______________________________________                                        Media for flasks studies                                                      Medium A    g/L        Medium B   g/L                                         ______________________________________                                        K.sub.2 HPO.sub.4                                                                         1          K.sub.2 HPO.sub.4                                                                        1                                           KCl         0.1        KCl        0.1                                         MgSO.sub.4.7H.sub.2 O                                                                     0.3        MgSO.sub.4.7H.sub.2 O                                                                    0.3                                         NaCl        0.1        NaCl       0.1                                         FeCl.sub.3  0.01       FeCl.sub.3 0.01                                        NaNO.sub.3  2.5        NaNO.sub.3 2.5                                         Sweet beet pulp                                                                           25         Lactose    5                                           Barley malt 15         Peptone    5                                           Wheat bran  5          pH         7.5                                         pH          7.5                                                               ______________________________________                                    

The strains were grown in shake flasks at 220 rpm and at 28° C. Samplesof each strain grown in Medium A were taken for analysis after 6 and 7days of culture. Samples of strains grown in Medium B were taken after 5days in culture. All samples were assayed for CMC-ase activity at pH 5and 7. The results of the CMC-ase assay are shown in Table 12.

                  TABLE 12                                                        ______________________________________                                        Cellulase production by different strains of Chrysosporium                           medium A  medium A    medium B                                                (6 days)  (7 days)    (5 days)                                                  CMC-ase       CMC-ase       CMC-ase                                  Strains  RS    pH 5   pH 7 RS  pH 5 pH 7 RS  pH 5 pH 7                        ______________________________________                                        1 VKMF 2117                                                                            2.7   0      0.46 2.6 0.00 0.00 2.3 0.21 0.09                        2. VKMF 2116                                                                           1.1   0.22   0.04 0.2 0.38 0.61 4.0 0.58 0.59                        3. VKMF 2121                                                                           1.9   0      0.57 1.1 0.25 0.10 2.5 0.25 0.09                        4. ATCC  3.4   0.33   1.40 1.9 1.85 0.11 3.0 1.10 0.06                        24782                                                                         5. ATCC  1.0   1.54   0.90 0.9 0.17 0.20 4.3 0.81 0.90                        34151                                                                         6. ATCC  4.4   0.21   0.49 2.0 0.68 0.34 2.5 1.29 0.06                        44006                                                                         7. VKMF 2119                                                                           4.1   0      0.08 2.7 0.29 0.00 3.8 0.95 0.04                        8. VKMF 2120                                                                           4.5   0      0.17 2.3 0.23 0.00 2.3 0.12 0.00                        9. VKMF 2875                                                                           1.6   0      1.01 1.7 0.00 0.00 3.8 1.96 0.05                        10. VKMF 2.4   0      0.03 0.8 0.22 0.00 5.0 0.43 0.00                        2877                                                                          11. C1   2.9   1.70   1.65 nt  nt   nt   0.1 0.89 0.80                        (VKMF                                                                         3500D)                                                                        ______________________________________                                         RS = concentration of reducing sugars in the fermentation medium at the       end of fermentation, g/L (NelsonSomogyi method).                              pH 5, pH 7 = the values of pH under which the CMCase activity of the          fermentation broth was assayed.                                               CMCase activity in U/ml.                                                      nt = not tested                                                          

In the cases of strains ATCC 34151 Chrysosporium pannorum, ATCCChrysosporium pruinosum, VKMF-2875 Chrysosporium keratinophilum, VKMF2116 Chrysosporium gueenslandicum the cell mass was separated bycentrifugation and cell free supernatant concentrated from 5 liters to0.5 liter by ultrafiltration using 10 kDa cut-off membrane. Then theultrafiltrated concentrate was lyophilized and stored for tests.

The following #-s of cellulase dry preparations were used:

47.2001-ATCC 34151 Chrysosporium pannorum,

47.2000-ATCC 24782 Chrysosporium pruinosum,

47.1001-VKMF-2875 Chrysosporium keratinophilum,

47.1000 - VKMF 2116 Chrysosporium gueenslandicum.

Protein content and activity fingerprints of these preparations aregiven in Table 4.

EXAMPLE 9 Stone Wash Tests

A. Tests with 2-L special washing machine

This system assesses the stone wash performance characteristics relatedto abrasion and backstaining using only small amounts of enzyme.

Desizing.

Forty pieces (30 g each, 25×20 cm) of denim fabric (roll) (1.2 kg) weredesized in a household washer at 60° C. for 20 minutes using afabric:liquor ratio of 1:6 (7.2 L) and 0.5 g/L (3.6 g) Sandoclean PCliquid (nonionic washing and wetting agent on base of ethyoxylated fattyalcohols with an average of 6 moles of ethylene oxide, 1 g/L (7.2 g)Sirrix 2UD (acidic buffered sequestration) and 1 g/L (7.2 g) Bactosol TKliquid (high temperature stable alpha-amylase) at a pH of about 5 to 6.After 20 minutes, the liquor was drained and the pieces washed for 5minutes with cold water (14 L) liquid ratio 1:10. The pieces were driedat 40° C. and used as a stock of comparable samples for thedetermination of cellulase activity

The cellulase treatment of the garment pieces was carried out in awashing machine consisting of an inner drum of 29 cm diameter drum--10.6l total volume (drum rotates at 20 rpm--five turns left--five turnsright). Each piece of fabric was sewn together with 4 rubber stoppersprior to the cellulase treatment to give a garment package that ensuredthat the mechanical effect occurred mainly on the darker outer side ofthe garment. Each drum was filled with one package and 10 additionalrubber stoppers.

The general wash conditions were: 30 g desized denim jean fabric,cellulase in 0.02M citrate buffer, 50° C., 60 minutes, garment:liquorratio 1:4. After the cellulase treatment the package was washed with hotwater (50° C.) (garment:liquid ratio 1:20) for 5 minutes and dried forevaluation.

Application trials were conducted using various C1 cellulasepreparations along with other cellulase preparations prepared fromdifferent species of Chrysosporium as well as the commercial NovoNordisk neutral cellulase products, Denimax XT (U.S. Pat. No. 4,435,307)and Ultra MG (WPO 91/17243). These application trials were set up toevaluate the stone wash performance characteristics of C-1 as well asseveral other species of Chrysosporium cellulases vs Novo's commercialneutral cellulases. The trials were run at neutral and alkaline pH's(6.5, 6.7, 7.0, and 8.0). The results are presented in Table 13.Garments treated with various C1 and other Chrysosporium cellulasepreparations showed similar wash performance characteristics to those ofthe commercial neutral cellulases Denimax XT and Denimax Ultra MG. TheC-1 and other Chrysosporium cellulase preparations showed good softeningeffect, bleaching/overall shade reduction, abrasion levels as well aslow backstaining values when run under neutral and alkaline pHconditions. Datacolor measurement is based on the degree of lightness ofthe sample (reflectance). The sample is exposed to white light (2 pulsedXenon flash lamps) and the remission is detected between 400 and 700 nmwith 16 diodes. Reflectance from the front side, the higher value themore abrasion. Reflectance from the back side, the higher value the morebackstaining.

                                      TABLE 13                                    __________________________________________________________________________    Enzyme Wash With Special 2 Liter Machine (135 grams of denim per run)                            CMCase                                                                              Endo (visc)                                                                           Liquor                                                                              Time   Datacolor                                                                          Datacolor                  Enzyme   Amount g                                                                           % OWG                                                                              U/g                                                                              /run                                                                             U/g                                                                              /run                                                                             °C.                                                                      ratio                                                                             pH                                                                              (min)                                                                            Buffer                                                                            Abrasion                                                                           Backstng                   __________________________________________________________________________    C-1 47.11.1                                                                            1.995                                                                              1.5  234                                                                              474                                                                              191                                                                              381                                                                              50                                                                              1:11                                                                              7.2                                                                             60 0.02MP                                                                            13.1 1.8                        Denimax XT                                                                             0.133                                                                              0.10 450                                                                              60 99 13 50                                                                              1:11                                                                              7.0                                                                             60 0.02MP                                                                            13.1 2.4                        C-1 47.12.1                                                                            2.100                                                                              1.5  167                                                                              338                                                                              138                                                                              290                                                                              50                                                                              1:11                                                                              6.7                                                                             60 0.02MP                                                                            14.2 1.8                        Denimax XT                                                                             0.420                                                                              0.30 450                                                                              182                                                                              99 42 50                                                                              1:11                                                                              6.6                                                                             60 0.02MP                                                                            14.0 2.3                        C-1 47.9.1                                                                             3.29 2.44 204                                                                              671                                                                              174                                                                              572                                                                              50                                                                              1:11                                                                              6.7                                                                             60 0.02MP                                                                            17.1 1.8                        C-1 47.16.1U                                                                           2.3  1.7  146                                                                              336                                                                              39 90 50                                                                              1:11                                                                              6.7                                                                             60 0.02MP                                                                            16.2 2.3                        47/1000.1                                                                              7.0  5.19 48 336                                                                              14 98 50                                                                              1:11                                                                              6.5                                                                             60 0.02MP                                                                            12.9 2.1                        47/2001.1                                                                              7.15 5.30 47 336                                                                              20 143                                                                              50                                                                              1:11                                                                              6.5                                                                             60 0.02MP                                                                            14.7 1.7                        Denimax UltraMG                                                                        0.132                                                                              0.10 134                                                                              18 243                                                                              32 50                                                                              1:11                                                                              7.3                                                                             60 0.02MP                                                                            14.1 3.5                        C-1 47.19.1                                                                            7.14 5.29 28 200                                                                              9  64 50                                                                              1:11                                                                              6.5                                                                             60 0.02MP                                                                            14.7 1.9                        C-1 47.0325                                                                            0.068                                                                              0.05 4965                                                                             338                                                                              964                                                                              66 50                                                                              1:11                                                                              7.0                                                                             60 0.02MP                                                                            15.1 2.3                        Denimax XT                                                                             1.0  0.74 450                                                                              450                                                                              99 99 50                                                                              1:11                                                                              6.5                                                                             60 0.02MP                                                                            19.1 2.8                        C-1 47.0528                                                                            0.08 0.05 4800                                                                             384                                                                              1782                                                                             143                                                                              50                                                                              1:11                                                                              6.5                                                                             60 0.02MP                                                                            18.5 3.0                        C-1 47.0325                                                                            0.136                                                                              0.10 4965                                                                             675                                                                              964                                                                              131                                                                              50                                                                              1:11                                                                              6.0                                                                             60 0.02MP                                                                            18.3 3.8                        C-1 47.0325                                                                            0.136                                                                              0.10 4965                                                                             675                                                                              964                                                                              131                                                                              50                                                                              1:11                                                                              7.0                                                                             60 0.02MP                                                                            18.9 3.2                        C-1 47.0325                                                                            0.136                                                                              0.10 4965                                                                             675                                                                              964                                                                              131                                                                              50                                                                              1:11                                                                              8.0                                                                             60 0.02MP                                                                            16.7 2.5                        Humicola 14.22.1                                                                       9.18 6.80 28 257                                                                              30 275                                                                              50                                                                              1:11                                                                              6.7                                                                             60 0.02MP                                                                            14.9 1.3                        Humicola 14.23.1                                                                       9.18 6.80 11 101                                                                              19 174                                                                              50                                                                              1:11                                                                              6.7                                                                             60 0.02MP                                                                            12.5 1.5                        T. reesei CP                                                                           0.30 0.22 9190                                                                             2737                                                                             2000                                                                             600                                                                              50                                                                              1:11                                                                              4.8                                                                             60 0.02CA                                                                            17.5 8.0                        Blank    0.00 0.00 0  0  0  0  50                                                                              1:11                                                                              6.5                                                                             60 0.02MP                                                                            9.1  n/a                        __________________________________________________________________________     0.02MP = Phosphate Buffer System                                              0.01CA = Citric Acid Buffer System                                            T. reesei CP = Commercial acid cellulase product produced from Trichoderm     reesei.                                                                       Datacolor Abrasion = reflectance from the front side, the higher the          values, the more abrasion, blank = 9.1                                        Datacolor Backstng = reflectance from the back side, the lower the values     the lower the back staining                                                   % OWG = for example for 1% OWG, 1 lb of enzyme is used on 100 lbs of          garment                                                                  

B. Tests with 35 lb washing machine

Application Trials were run in a 35 lb washing machine (35 lb washingmachine brand is Milnor--washer RPM is 30). Load size is 2400 g (3garments), garments used are Levi's 505 jeans. Water level for cellulasebath is 15 L for a liquor ratio of 6.25:1 (low). The water level for allother baths is 24 L for a liquor ratio of 10:1 (Med). The bufferingsystem used is MAP--monoammonium phosphate and DAP--diammonium phosphateto maintain the pH of 6.7 during the cellulase bath. In Trials 4, 5, 6 &7 a nonionic detergent was added to the cellulase bath, it is known thatadding a detergent to the cellulase bath will help in reducing thebackstaining on the garments. Zeke is a desizing product. SSCE isSuperscour, a nonionic detergent (Zeke and Super Scour are commercialspecialty textile chemical products offered by CPN International, Ltd.,Inc of Jupiter, Fla.). One Example of the Wash Formulas used in thesetrials is Trial 2. below;

    __________________________________________________________________________    Wash Formula - Trial 2.                                                       (C-1 47.0523)                                                                 Load (g) 2400 (3 gmts) Fabric: Denim Formula Time: 1:30                       Machine: 35# Milnor Weight: 14.5 oz Developed by:                             Step                                                                             Operation                                                                            Time (min)                                                                          Level                                                                            Temp (F.)                                                                          Chemical                                                                           Amount                                                                            % OWG                                                                              pH                                      __________________________________________________________________________     1 Desize 10    Med                                                                              150  Zeke 48 g                                                                              2                                             2 Drain Balance                                                               3 Rinse  2     Med                                                                              140                                                         4 Drain Balance                                                               5 Rinse  2     Med                                                                              130                                                         6 Drain Balance                                                               7 Abrasion                                                                             75    Low                                                                              125  MAP  29 g                                                                              buffer                                                                             67                                                      (15L)                                                                                 DAP  10 g                                                                     C-1  1.2 g                                                                             0.05                                          8 Drain Balance                                                               9 Wash   10    Med                                                                              160  SSCE 24 g                                                                              1                                            10 Drain Balance                                                              11 Rinse  3     Med                                                                              120                                                        12 Drain Balance                                                              13 Rinse  3     Med                                                                              100                                                        14 Drain Balance                                                              15 Rinse  3     Med                                                                              100                                                        16 Drain Balance                                                              17 Extract                                                                              2                                                                   __________________________________________________________________________

In the example above, and in commercial use, one skilled in the art willappreciate that the use of pumice stones in the stonewash process willenhance the stonewash effect on the garments.

The results in Table 14. show that the C1 cellulase preparations#47.0325 and #47.0528 performed better in terms of the overall level ofabrasion achieved on the garments and well within the range of thebackstaining level of the other commercial neutral cellulase productstested.

                  TABLE 14                                                        ______________________________________                                        Comparison of C-1 Cellulase Preparations 47.0325 & 47.0528                    To Commercial Neutral Cellulases Denimax XT & BTU 202 - 318                   (which contains Denimax XT)                                                   ______________________________________                                                        %                                                             Trial                                                                              Cellulase  OWG    Wt(g)                                                                              Detergent                                                                            T(°F.)                                                                       pH  t(min)                           ______________________________________                                        1    Denimax XT 0.50   12.0 no     130   6.7 75                               2    C-1 47.0528                                                                              0.05   1.2  no     125   6.7 75                               3    C-1 47.0325                                                                              0.10   2.4  no     125   6.7 75                               4    Denimax XT 0.50   12.0 yes    130   6.7 75                               5    C-1 47.0528                                                                              0.05   1.2  yes    125   6.7 75                               6    C-1 47.0325                                                                              0.10   2.4  yes    125   6.7 75                               7    BTU 202-318                                                                              2.50   60.0 yes    130   SB  75                               ______________________________________                                        ABRASION           BACKSTAINING                                               (Most to Least)    (Least to Most)                                            ______________________________________                                        Trial 5            Trial 4                                                    Trial 6            Trial 5                                                    Trial 2            Trial 6                                                    Trial 3            Trial 3                                                    Trial 4            Trial 1                                                    Trial 1            Trial 2                                                    Trial 7            Trial 7                                                    ______________________________________                                         Figure Legand for Table 14                                                    All of the trials in table 14 were cleaned up with Super Scour (nonionic      detergent) at 1.0% OWG, 160 F. for 5 minutes. SB = Self Buffered  the         commercial product "ROCKSOFT" BTU 202318 contains Denimax XT, detergent       and a buffer system as well as other additives to help enhance the stone      wash performance of this commercial product.                             

C. Tests with 60-L special washing machine

Whole denim garments were desized as described for the 2-L washingmachine tests. Each wash test was made with 1 pair of jeans (700 g), 2.8L liquid (fabric:liquid ratio 1:4). All jeans were from the same dyelot. They were prewashed using an oxidation method for 15 minutes, thendried. Blue jeans washed at neutral pH with formulated C1 cellulasepreparations 47.0325 using 2.4 grams per trial and 47.0528 using 1.5grams for one trial and using 1.0 gram for a second trial were compareddirectly against blue jeans washed under neutral pH conditions andsimilar formulations using Denimax XT at 12 grams per trial and twoother commercial neutral cellulases; Bactosol JE using 2.0% OWG and BTU202-318 using 2.0% OWG (Bactosol JE and BTU 202-318 contain Denimax XT,buffer, detergent as well as other additives to enhance their washperformance). Table 15. shows that the blue jeans from all three C-1trials outperformed the three commercial neutral cellulase products interms of the level of abrasion achieved as well as the overall colorreduction of the garments. The level of backstaining on the blue jeansfrom all six trials was very good, they were very similar to one anotherand what one would expect and see when using Novo's neutral cellulaseDenimax XT. The backstaining values for all three of these C-1 trialswere within the range of the backstaining values as shown in Table 13.The finished garments from these trials and the trials as rated andshown in Table 14 above were rated in a blind study by four independentgroups, of three or more people per group. The people that made up eachof these groups are considered to be skilled in the art of stonewashing.They were asked to place each of the garments in the following order:(1) Greatest overall abrasion and color reduction to least overallabrasion and color reduction; and (2) Backstaining, lowest level ofbackstaining to highest level of backstaining (See Table 15).

                                      TABLE 15                                    __________________________________________________________________________                                 ABRASION/                                                                     COLOR                                            TRIAL                                                                             ENZYME DOSAGE                                                                              BUFFER                                                                             DETERGENT                                                                            REDUCTION                                                                            BACKSTAINING                              __________________________________________________________________________    Trial A                                                                           C-1 47.0528                                                                          1.5 grams                                                                           Phosphate                                                                          Yes    ++++++ 5                                         Trial B                                                                           C-1 47.0528                                                                          1.0 grams                                                                           Phosphate                                                                          Yes    +++++  2                                         Trial C                                                                           C-1 47.0325                                                                          2.4 grams                                                                           Phosphate                                                                          Yes    +++++  4                                         Trial D                                                                           Denimax XT                                                                           12.0 grams                                                                          Phosphate                                                                          Yes    ++++   3                                         Trial E                                                                           BTU 202-318                                                                          2.0% OWG                                                                            Phosphate                                                                          Yes    +++    6                                         Trial F                                                                           Bactosol JE                                                                          2.0% OWG                                                                            Citrate                                                                            Yes    +++    1                                         __________________________________________________________________________     Legend for Table 15:                                                          Abrasion/Color Reduction -- ++++++ (+6) best (=>++++ (4) is considered        good and was comparable to commercial netural cellulases (e.g.  Denimax       XT)                                                                           Backstaining -- The lower number the better (all jeans were judged to be      within the range of backstaining as found when using Novo's Denimax TX).      Neutral cellulase significantly decreased backstaining compared with          traditional acid cellulases such as Trichoderma (see Example 13)              % OWG -- % Of Weight of Garment, for example for 100 lbs of jeans             dryweight at 1% OWG, 1 lb of enzyme is used.                             

D. Light reflectance

Another test to evaluate backstaining is to measure the lightreflectance of a treated fabric. At the end of washing treatment, jeanssamples were analyzed using a reflectometer at two differentwavelengths: (1) the higher the signal detected at 680 nm (measured atthe outside of the jeans), the lower the backstaining; and (2) thehigher the signal detected at 420 nm (measured at the inside of thejeans), the lower the backstaining. Table 16. compares the reflectancevalues of denim jeans after treatment with commercial cellulases fromNovo Nordisk and Genencor International to C-1 preparation #47.6.1.

                  TABLE 16                                                        ______________________________________                                        Enzyme                 680 nm  420 nm                                         ______________________________________                                        Denimax L (neutral cellulase, Novo)                                                                  23      20                                             Primafast 100 (acid cellulase, Genencor)                                                             20      13                                             C1 47.6.1 (neutral/alkaline cellulase)                                                               22      18                                             ______________________________________                                    

The light reflectance values for the C1 cellulase were similar to thoseobtained with Novo Nordisk's commercial product Denimax L, a neutralcellulase, at both 680 and 420 nm and the light reflectance values forC1 cellulase were significantly better than those obtained withGenecor's commercial product Primafast 100, a acid cellulase, at both680 and 420 nm.

E. Tests in semi-industrial washing machine

Test #1.

2 Jeans, weight 1343 gr

Water ratio 6:1

pH 5.5

Temp. 54° C.

Enzyme: C1 (preparation #47.6.1) 12 gr (0.9%)

Abrasion time 90 minutes

Drop bath

Rinse 5 minutes with 1% non-ionic detergent at 66° C.

Drop bath

Rinse cold

Drop bath

Soften for 5 minutes with cationic softener at 49° C.

Extract and dry.

Test #2.The same procedure as Test #1, above, except Denimax 700 T (2%OWG 28.9 gr) enzyme was used and wash conditions were conducted at pH7.0, 54° C.

C1 cellulase was compared to Denimax 700 T, a neutral cellulasecommercial product made by Novo. All jeans were from the same dye lot.They were prewashed for 15 minutes using an oxidation method then dried.

The jeans treated with C1 cellulase preparation #47.6.1 showed slightlyless abrasion and lower backstaining than the jeans treated with Denimax700T cellulase.

EXAMPLE 10 C1 Cellulase as an Additive to Laundry Detergent

A. Soil Release from Cotton

Wash performance of C1 cellulase preparation #47.9.1 was tested usingthe wash-performance procedure PW 9406 (Solvay). Soil (ink) release fromcotton fabric was tested by Delta Reflectance (%). Wash test compared aC1 cellulase preparation (#47.9.1) to Celluzyme 0.7 T from Novo Nordiskin the presence and absence of alkaline protease Opticlean L500. Theresults of this test are shown in the Table 17.

C1 cellulase has soil release properties from ink soiled cotton atneutral pH in a color type detergent as the cellulase enzyme fromHumicola insolens.

                  TABLE 17                                                        ______________________________________                                        Detergent wash test with C1 cellulase(*)                                                     CMC-ase Reflectance Data (%)                                   Enzyme tested (pH 7.0)                                                                         dosage (U/I)                                                                            1        2                                         ______________________________________                                        Cellulzyme 0.7 T 200       3.68     4.75                                      Celluzyme 0.7 T  500       2.68     4.07                                      Celluzyme 0.7 T + 5000 DU/1 (**)                                                               200       2.07     3.13                                      Celluzyme 0.7 T + 5000 DU/1 (**)                                                               500       2.08     3.22                                      C1 #47.9.1       200       2.18     2.88                                      C1 #47.9.1.      500       2.77     3.72                                      C1 #47.9.1 + 5000 DU/1 (**)                                                                    200       1.15     1.91                                      C1 #47.9.1 + 5000 DU/1 (**)                                                                    500       2.81     3.30                                      None (control)   none      0        0                                         ______________________________________                                         AADU = Du = Delft unit, Du/1 = Defft unit per liter                           (*) 40 C.°, 45 min, drying at 68° C., 75 min                    (**) Alkaline protease Opticlean L500                                    

B. The Stability of C1 Cellulase with Serine Proteases

As serine proteases, a trypsin (3.2 μM, from Bovine Pancreas, activity10,000-13,000 N-benzyl-L-argine ethylester (BAEE)/mg, Sigma T-8253) andan α-chymotrypsin (8 μM, from Bovine Pancreas, 40-60 U/mg, Sigma C-4129)were used.

The proteases were incubated with C1 cellulase at 20° C. and pH 7.0.Chymotrypsin did not decrease C1 activity for 12 hours and trypsin ledto a slight decrease (around 20%) of C1 activity, see Table 18.

Trypsin and chymotrypsin did not significantly change the stability ofC1 CMC-ase at pH-s 4.5 and 7.0 at 50° and 57° C., see Table 18.

                  TABLE 18                                                        ______________________________________                                        The effect of proteases on CMC-ase activity of C1 cellulase (#47.9.1)                   Temperature      Incubation                                                                            CMC-ase activity                           Protease  (°C.)                                                                             pH    time (h)                                                                              remaining (%)                              ______________________________________                                        None (control)                                                                          20         7.0   12      70                                         + Chymotrypsin                                                                          20         7.0   12      70                                         + Trypsin 20         7.0   12      50                                         None (control)                                                                          50         4.5   3       100                                        + Chymotrypsin                                                                          50         4.5   3       100                                        + Trypsin 50         4.5   3       100                                        None (control)                                                                          50         7.0   3       78                                         + Chymotrypsin                                                                          50         7.0   3       60                                         + Trypsin 50         7.0   3       68                                         None (control)                                                                          57         4.5   3       62                                         + Chymotrypsin                                                                          57         4.5   3       60                                         + Trypsin 57         4.5   3       62                                         None (control)                                                                          57         7.0   3       30                                         + Chymotrypsin                                                                          57         7.0   3       30                                         + Trypsin 57         7.0   3       30                                         ______________________________________                                    

C. The Effect of Citrate, EDTA Tween-80 and Persulfate on CMC-aseactivity

Changing from acetate to citrate buffer (a chelating agent) did noteffect the of C1 CMC-ase activity (molarity of buffers -0.1M, pH 4.5, 50and 57° C.), see Table 19.

EDTA (Ethylene Diamine Tetraacetic Acid) (5 mM) as a chelating agent atpH 4.5 and 50° C. did not change CMC-ase activity. At pH 4.5 (57° C.)and at pH 7.0 (50° C.) EDTA caused slight decreases in CMC-ase activity.At pH 7.0 and 57° C., EDTA caused slight increase in CMC-ase activity,see Table 19.

Non-ionic detergent Tween-80 (3 g/L, polyoxyethylene sorbitanemonooleate), did not change CMC-ase activity of C1 (at pH-s 4.5 and 7.0and at 50° and 57° C., see Table 19.

Oxidizing agent persulfate (3 g/L) did not change CMC-ase activity of C1(at pH-s 4.5 and 7.0 and at 50° and 57° C.), see Table 19.

C1 CMC-ase is resistant to serine proteases (trypsin and chymotrypsin),chelating agents (EDTA, citrate), non-ionic detergent (Tween-80) and tooxidizing agent (persulfate).

                  TABLE 19                                                        ______________________________________                                        The effect of citrate, EDTA, Tween-80 and persulfate on activity of           C1 cellulasese (#47.9. 1). Incubation time - 3 hours.                                             Temperature    CMC-ase activity                           Effector Concentration                                                                            (°C.)                                                                             pH  remaining (%)                              ______________________________________                                        None (control)                                                                         --         50         4.5 100                                        Citrate  0.1 M      50         4.5 100                                        EDTA     5 mM       50         4.5 100                                        Tween-80 3 g/L      50         4.5 100                                        Persulfate                                                                             3 g/L      50         4.5 97                                         None (control)                                                                         --         57         4.5 62                                         Citrate  0.1 M      57         4.5 65                                         EDTA     5 mM       57         4.5 60                                         Tween-80 3 g/L      57         4.5 68                                         Persulfate                                                                             3 g/L      57         4.5 65                                         None (control)                                                                         --         50         7.0 78                                         EDTA     5 mM       50         7.0 50                                         Tween-80 3 g/L      50         7.0 52                                         Persulfate                                                                             3 g/L      50         7.0 50                                         None (control)                                                                         --         57         7.0 30                                         EDTA     5 mM       57         7.0 38                                         Tween-80 3 g/L      57         7.0 25                                         Persulfate                                                                             3 g/L      57         7.0 30                                         ______________________________________                                    

EXAMPLE 11 Stone wash tests of cellulase samples produced by differentstrains of Chrysporium

Preparations #-s 47.1000, 47.1001, 47.2000 and 47.2001 produced bydifferent strains of Chrysosporium were used for wash test with 2-Lspecial wash machine at pH 6.5, 50° C., during 60 min with 135 g ofdesized denim jean fabric. Total amount of CMC-ase activity per trialwas constant and equal to 336 U/run. After drying abrasion andbackstaining of garment was evaluated by Datacolor measurement. Theresults are presented in Table 20. The results show that cellulasesproduced from different strains of Chrysosporium demonstrate similarwash performance at neutral pH in terms of abrasion and backstaininglevels to the cellulases produced by the C-1 species Chrysosporiumlucknowense Garg 1966.

                  TABLE 20                                                        ______________________________________                                        Stone wash activity of cellulase preparations from different strains of       Chrysosporium                                                                 Preparation #                                                                              Abrasion (*)                                                                            Backstaining (**)                                      ______________________________________                                        47.1000      12.3      1.6                                                    47.1001      12.1      1.6                                                    47.2000      14.2      1.7                                                    47.2001      14.6      1.6                                                    ______________________________________                                         (*) -- reflectance from front side, the higher value the more abrasion,       blank = 9.1                                                                   (**) -- reflectance from back side, the higher value the more backstainin                                                                              

EXAMPLE 12 Purification of Cellulase Components

1. Selection of the C1 samples for purification. The C1 cellulasepreparation #47.11.1 was chosen for further purification in view of thefact that 47.11.1 possessed (i) high protein content; (ii) high FPA andCMC-ase activity (see Table 4).

2. Isolation and purification of C1 complex component. The firstpurification step included ion exchange chromatography on DEAE-SpheronC-1000 (Chemapol, Czech Republic). Dry C1 cellulose preparation (1.5 g)was dissolved in 15 mL of 0.01M Na-phosphate buffer, pH 7. The solutionwas centrifuged and the supernatant desalted using an Acrylex P-2column. The desalted sample was then applied to the DEAE-Spheron column(2.5×40 cm) in 0.01M phosphate buffer, pH 7. Flow rate was 1 mL/minute.Fraction I was eluted in the start buffer. Fraction II was eluted acrossa 0 to 1M NaCl gradient. Fraction III was eluted in 1M NaCl after thegradient was completed. All fractions possessed cellulolytic activities.Fraction III contained pigment because separation from pigment did notoccur using DEAE-Spheron chromatography. Fractions I, II, and III werelyophilized to concentrate and preserve enzyme activity.

Fraction II was further purified by ion exchange chromatography on "MONOQ" "MONOBEADS" (Pharmacia Biotech, Uppsala, Sweden). "MONO Q" is astrong anion exchange resin which binds negatively charged molecules.300 mg of Fraction II was dissolved in 3 mL of 0.01M Na-phosphatebuffer, pH, 7, desalted, and applied on a "MONO Q" column (1.5×10 cm) in0.01M phosphate starting buffer. Fraction II-1 was eluted in startbuffer, and Fraction II-2 was eluted across a 0 to 1M NaCl gradient at aflow rate of 1 mL/minute. Both fractions showed cellulolytic activities.Fractions II-1 and II-2 were lyophilized.

3. SDS-PAGE of protein fractions. Gel electrophoresis was carried out in10% separating polyacrylamide gel under denaturing conditions. FractionI has two main protein bands (around 30 and 40 kDa), Fraction II-1 has6-7 main bands (between 35-70 kDa), Fraction II-2 has three main bands(around 40, 50, and 60 kDa), Fraction III contains numerous bands andresembles the SDS-PAGE picture of the unfractionated C1 complex.Reagents and kits from Bio-Rad (U.S.A.) were used for SDS-PAGE plates(100×8×0.75 mm). Coomasee brilliant blue R-250 in 25% trichloraceticacid was used for protein staining.

4. pH-dependencies of CMC-ase activity of protein fractions. Table 21represents the pH dependencies of CMC-ase activity of partially purifiedfractions of C1 cellulase. CMC-ase activity was measured at 50° C. (CMCwas hydrolyzed for 10 minutes). To create the different pH ranges, thefollowing buffer systems were used: acetate buffer (pH 4-5), phosphatebuffer (pH 6-8), and carbonate buffer (pH 8.5-10). In addition, auniversal buffer system was used which consisted of acetate, borate, andphosphate (pH 4-10).

                  TABLE 21                                                        ______________________________________                                        The effect of pH on CMC-ase activity of C1 protein fractions                  (50° C.).                                                              CMC-ase activity (%)                                                          pH    Fraction I    Fraction II-1                                                                           Fraction III                                    ______________________________________                                        4.0   65            65        100                                             4.5   90            85        100                                             5.0   100           100       95                                              5.5   90            100       90                                              6.0   65            90        85                                              6.5   60            85        75                                              7.0   40            65        54                                              7.5   15            50        35                                              8.0    5            35        32                                              8.5   35            25        10                                              9.0    8            20        15                                              ______________________________________                                    

pH-dependency of Fraction I after DEAE-Spheron had maximum under pH 5.0and 8.5. Fraction II-1 (after DEAE-Spheron and "MONO Q" ion exchangechromatography) had a non-symmetric bell-type pH profile with a maximumat pH 5-6.5 and with a "shoulder" at pH 7-9 and a pH profile more orless the same as the pH profile of the unfractionated C1 preparation(see Tables 21. and Table 7.). Fraction III after DEAE-Spheron had a pHhad a pH-optimum 4-5.5 with a "shoulder" at pH6-9.

5. Stability of CMC-ase of protein Fraction I (after DEAE-Spheron).Table 22. shows temporal CMC-ase activity curves of Fraction I afterDEAE-Spheron ion exchange chromatography at different pH (5.2-8.7) and50° C. CMC-ase activity of Fraction I was most stable at pH 5.2-7.2(around 35% to 45% of activity remained after 3 hours). At pH 7.7, 60%of activity was lost after about 1 hour, whereas at pH 8.3 and 8.7, 50%of activity was lost after about 0.5 hour. At pH 8.3, 100% of CMC-aseactivity was lost after 3 hours, and at pH 8.7, 100% of activity waslost after 2 hours.

                  TABLE 22                                                        ______________________________________                                        Stability of CMC-ase of a protein fraction I after DEAE-Spheron at            50° C.                                                                 CMC-ase activity remained (%)                                                 Time (h)                                                                             pH 5.2    pH 7.2  pH 7.7  pH 8.3                                                                              pH 8.7                                 ______________________________________                                        0      100       100     100     100   100                                    0.5    97        75      65      50    50                                     1      90        55      40      25    15                                     2      40        50      35      10     0                                     3      30        45      25       0     0                                     ______________________________________                                    

EXAMPLE 13 C-1 Cellulase Production in 60 Liter Batch Fermentor

1. Inoculum Preparation

Inoculum preparations or starter cultures for the batch fermentationwere prepared as follows. One milliliter (1 ml) of C-1 spore culture wasused to inoculate each of two flasks to generate a total of 2.0 litersof inoculum. The starter culture was incubated at 150 rpm, at 30° C. for56 hours.

    ______________________________________                                        Medium for Inoculum Preparation*                                              ______________________________________                                        K.sub.2 HPO.sub.4 0.5         g/L                                             MgSO.sub.4.7H.sub.2 O                                                                           0.15                                                        KCl               0.05                                                        FeSO.sub.4.7H.sub.2 O                                                                           0.007                                                       yeast extract (ohly KAT)                                                                        1.0                                                         peptone (Hormel PSR 5)                                                                          10.0                                                        lactose           10.0                                                        glucose           10.0                                                        ______________________________________                                         *The pH of the inoculum medium was adjusted to pH 7.0 with NaOH, the medi     was then autoclaved for 35 minutes at 121° C. in two six liter         baffled flasks each containing one liter of medium.                      

2. Cellulase Production in 60 Liter Batch Fermenter (Preparation of47.0325)

The two liter shaker flask inoculum culture prepared above, was usedinoculate 40 liters of medium contained in a 60 liter fermenter. Themedium for fermentation was as follows:

    ______________________________________                                        Fermentation Medium*                                                          ______________________________________                                        K.sub.2 HPO.sub.4 0.22       g/L                                              KH.sub.2 PO.sub.4 0.08       g/L                                              (NH.sub.4).sub.2 SO.sub.4                                                                       4.0        g/L                                              Na.sub.3 citrate.2H.sub.2 O                                                                     4.0        g/L                                              MgSO.sub.4.7H.sub.2 O                                                                           0.03       g/L                                              CaCl.sub.2.2H.sub.2 O                                                                           0.4        g/L                                              FeSO.sub.4.7H.sub.2 O                                                                           0.5        mg/L                                             MnSO.sub.4.7H.sub.2 O                                                                           0.5        mg/L                                             ZnSO.sub.4.7H.sub.2 O                                                                           0.2        mg/L                                             CoCl.sub.2.6H.sub.2 O                                                                           0.24       mg/L                                             lactose           5.0        g/L                                              yeast extract (ohly KAT)                                                                        0.05       g/L                                              defatted cotton seed flour                                                                      5.0        g/L                                              (Pharmamedia)                                                                 cellulose (Signmacell 50)                                                                       20.0       g/L                                              pH                7.0                                                         ______________________________________                                         *The 40 liters medium was in deionized water, and was sterilized for 45       minutes at 121° C.                                                

After inoculation of the fermentation medium, pH was maintained above6.9 by addition of NH₃ and below 7.1 by addition of H₂ SO₄. Thefermenter was incubated for 64 hours with agitation and aeration asnecessary to maintain dissolved oxygen greater than 30% of saturation.

3. Recovery of Cellulase Activity

Suspended solids from the fermented culture were removed by filtrationon large Buchner funnel using Whatman 54 filter paper and 10 g/L Celite503 as filter aid. The filtrate was collected, and the cellulaseconcentrated by ultrafiltration using 10,000 MW cutoff hollow fiberfilter. The concentrate was freeze dried. The dried concentrate wasdesignated cellulase preparation 47.0325. The activity of thispreperation is given in Table 4.

EXAMPLE 14 Mutation procedure used to generate mutant stain of C-1

A spore suspension was prepared using a Pridham agar plate (4 g/L yeastextract, 10 g/L malt extract, 10 g/L glucose, 15 g/L agar) containing asporulated culture of strain C1. The plate was flooded with 10 ml of0.05% Tween 80. The suspension was transferred to a sterile screw captube and vortexed on high for 1 minute. The suspension was then filteredthrough a column to remove mycelium. Spores were counted and diluted to7×10⁵ spores per ml in water. Ten mls of the spore suspension weretransferred to a standard glass petri dish. The spores were irradiatedfor 75 seconds at 720 μWatts/cm² using a Pen-Ray UV bulb. The sporesuspension was gently stirred throughout the irradiation using a sterilepaper clip as a magnetic stir bar. Following irradiation, the sporesuspension was taken to a foil wrapped tube, diluted in water and platedin dim light to NH₄ minimal medium as defined below. After incubating 20days at 30 degrees C., a colony was identified as a large colony with alarge zone of cellulose clearing around the colony.

NH₄ Minimal Medium, pH 7.5

1 g/L K₂ HPO4₄

0.1 g/L KCl

0.3 g/L MgSO₄ •7H₂ O

0.1 g/L NaCl

16 mg/L FeCl₃ •6H₂ O

1.92 g/L(NH₄)₂ SO₄

15 g/L Difco Noble agar

2.5 g/L acid swollen cellulose (added as a 1.25% stock afterautoclaving)

0.5 g/L sodium deoxycholate (added after autoclaving)

EXAMPLE 15 C-1 Mutant Cellulase Production in 60 Liter BatchFermentation Flasks (Preparation Of 47.0528)

1. Inoculum Preparation

Preparations of starter cultures for the fed batch fermentation wereprepared as described in Example 13 (section 1).

2. Cellulase Production In 60 Liter Batch Fermentation

The two liter inoculum was used to inoculate 40 liters of fermentationmedium as described below.

    ______________________________________                                        Fermentation Medium*                                                          ______________________________________                                        K.sub.2 HPO.sub.4 0.44       g/L                                              KH.sub.2 PO.sub.4 0.16       g/L                                              (NH.sub.4).sub.2 SO.sub.4                                                                       3.0        g/L                                              Na.sub.2 citrate.2H.sub.2 O                                                                     4.0        g/L                                              MgSO.sub.4.7H.sub.2 O                                                                           0.06       g/L                                              CaCl.sub.2.2H.sub.2 O                                                                           0.8        g/L                                              FeSO.sub.4.7H.sub.2 O                                                                           0.1        mg/L                                             MnSO.sub.4.7H.sub.2 O                                                                           0.04       mg/L                                             ZnSO.sub.4.7H.sub.2 O                                                                           0.04       mg/L                                             CoSO.sub.4.6H.sub.2 O                                                                           0.048      mg/L                                             lactose           5.0        g/L                                              yeast extract (ohly KAT)                                                                        0.1        g/L                                              defatted cotton seed flour                                                                      10.0       g/L                                              (Pharmamedia)                                                                 cellulose (Sigmacell 50)                                                                        20.0       g/L                                              ______________________________________                                         *The 40 liters medium was in deionized water, and sterilized by               autoclaving for 45 minutes at 121° C.                             

3. Fermentation Conditions

The pH was maintained at around 7.0 and controlled by addition of NH₃ atpH above 6.9, and addition of H₂ SO₄ at pH below 7.1. Incubation timewas 87 hours, agitation and aeration were as necessary to maintaindissolved oxygen greater than 30% of saturation. At 40 hours, 3.0 litersof feed solution as described below, was added at a rate of 5.0 ml each5 minutes.

    ______________________________________                                        Feed Solution for Fermenter                                                   ______________________________________                                        K.sub.2 HPO.sub.4 0.88       g/L                                              KH.sub.2 PO.sub.4 0.32       g/L                                              (NH.sub.4).sub.2 SO.sub.4                                                                       4.0        g/L                                              Na.sub.2 citrate.2H.sub.2 O                                                                     4.0        g/L                                              MgSO.sub.4.7H.sub.2 O                                                                           0.12       g/L                                              CaCl.sub.2.2H.sub.2 O                                                                           0.16       g/L                                              FeSO.sub.4.7H.sub.2 O                                                                           0.2        mg/L                                             MnSO.sub.4.7H.sub.2 O                                                                           0.08       mg/L                                             ZnSO.sub.4.7H.sub.2 O                                                                           0.08       mg/L                                             CoCl.sub.2.6H.sub.2 O                                                                           0.096      mg/L                                             lactose           20.0       g/L                                              yeast extract (ohly KAT)                                                                        0.2        g/L                                              Pharmamedia       20.0       g/L                                              cellulose (Sigmacell 50)                                                                        20.0       g/L                                              ______________________________________                                    

4. Recovery of Cellulase Activity

Suspended solids were removed by filtration on large Buchner funnelusing Whatman 54 filter paper and 10 g/L Celite 503 as filter aid. Thefiltrate was collected and cellulase concentrated by ultrafiltrationusing 10,000MW cutoff hollow fiber filter. The concentrate was dried byfreeze-drying. The concentrate was designated cellulase preparation47.0528 (activity is given in Table 4).

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

We claim:
 1. A neutral and/or alkaline cellulase comprise obtained by amethod which comprises:(a) growing a wild type Chrysosporium lucknowenseculture on a suitable medium; and (b) isolating said neutral and/oralkaline cellulase composition from said Chrysosporium lucknowenseculture.
 2. The composition of claim 1, wherein said Chrysosporiumlucknowense is Chrysosporium lucknowense Garg 27K, accession number VKMF-3500D.
 3. A composition for the enzymatic treatment of cellulosicfibers or fabrics, comprising a cellulase whose amino acid sequence isencoded by a nucleic acid sequence from a wild type fungus of the genusChrysoporium, said compostion having a pH between about 8.0 and about12.0.
 4. The composition of claim 3, wherein the fungus is selected fromthe group consisting of Chyrosoporium lucknowense, Chrysosporiumpannorum, Chrysosporium pruinosum, Chrysoporium keratinophilum,Chrysosporium lobatum, Chrusoporium merdarium, Chrusoporiumqueenslandicum, and Chrysosporium tropictum.
 5. The composition of claim4, wherein the fungus is Chrysosporium lucknowense.
 6. The compositionof claim 3, wherein the cellulase is isolated or obtained form awild-type fungus of the genus Chrysosporium.
 7. The composition of claim4, wherein the cellulase is isolated or obtained from a wild-type fungusselected from the group consisting of Chrysosporium lucknowense,Chrysosporium pannorum, Chrysosporium pruinosum, Chrysosporiumkeratinophilum, Chrysosporium lobatum, Chrysosporium merdarium,Chrysosporium queenlandicum and Chrysosporium tropicum.
 8. Thecomposition of claim 5, wherein the cellulase is isolated or obtainedfrom a wild-type fungus Chrysosporium lucknowense.
 9. A composition asdescribed in any one of claims 3-8 further comprising one or morecomponents that enhance the performance of the cellulase, wherein thecomponents are selected from the group consisting of pumice stones,abrasives, softeners, solvents, preservatives, bleaching agents, bluingagents, flourescent dyes, antioxidants, solubilizers, detergents,surfactants, enzymes, builders, anti-redoposition agents, buffers,caking inhibitors, masking agents for factors inhibiting the cellulaseactivity, and cellulase activators.
 10. A composition as described inany one of claims 3-8, wherein the pH is between 10.0 and about 11.0.11. The composition of claim 9, wherein the pH is between 10.0 and about11.0.
 12. A composition for the enzymatic treatment of cellulosic fibersof fabrics, comprising a neutral and/or alkaline cellulose whose aminoacid sequence is encoded by a nucleic acid sequence from a wild-typefungus of thegenus Chrysosporium, said composition further comprisingone or more components that enhance the performance of the cellulase,wherein the components are selected from the group consisting of addedproteases, detergents, and surfactants.
 13. The composition of claim 12,wherein the fungus is selected from the group consisting ofChrysosporium lucknowense, Chrysosporium pannorum, Chrysosporiumpruinosum, Chrysosporium keratinophilum, Chrysosporium lobatum,Chrysosporium merdarium, Chrysosporium queenlandicum, and Chrysosporiumtropicum.
 14. The composition of claim 13, wherein the fungus isChrysosporium luckowense.
 15. The composition of claim 12, wherein thecellulase is isolated or obtained from a wild-type fungus of the genusChrysosporium.
 16. The composition of claim 13, wherein the cellulase isisolated or obtained from a wild-type selected from the group consistingof Chrysosporium lucknowense, Chrysosporium pannorum, Chrysosporiumpruinosum, Chrysosporium keratinophilum, Chrysosporium lobatum,Chrysosporium merdarium, Chrysosporium queenlandicum, and Chrysosporiumtropicum.
 17. The composition of claim 14, wherein the cellulase isisolated or obtained from a wild-type fungus Chrysosporium lucknowense.18. A composition for the enzymatic treatment of cellulosic fibers orfabrics, having from about 33 to 191 units of endo-1,4-β-glucanaseactivity per gram of dry composition, as measured by an endoviscometricassay, of a neutral and/or alkaline cellulose whose amino acid sequenceis encoded by a nucleic acid sequence from a wild-type fungus of thegenus Chrysosporium.
 19. The composition of claim 18, wherein the fungusis selected from the group consisting of Chrysosporium luckowense,Chrysosporium pannorum, Chrysosporium keratinophilum, Chrysosporiumlobatum, Chrysosporium merdarium, Chrysosporium queenslandicum, andChrysosporium tropicum.
 20. The composition of claim 19, wherein thefungus is Chrysosporium lucknowense.
 21. A composition for the enzymatictreatment of cellulosic fibers or fabrics, having from about 33 to 191units of endo-1,4-β-glucanase activity per gram of dry composition, asmeasured by an endoviscometric assay, of a neutral and/or alkalinecellulose isolated or obtained from a wild-type fungus of the genusChrysosporium.
 22. The composition of claim 21, wherein the fungus isselected from the group consisting of Chrysosporium luckowense,Chrysosporium pannorum, Chrysosporium keratinophilum, Chrysosporiumlobatum, Chrysosporium merdarium, Chrysosporium queenslandicum, andChrysosporium tropicum.
 23. The composition of claim 22, wherein thefungus is Chrysosporium lucknowense.
 24. A composition for the enzymatictreatment of cellulosic fibers or fabrics, having about 120 to 191 unitsof endo-1,4-β-glucanase activity per gram of dry composition, asmeasured by an endoviscometric assay, of a neutral and/or alkalinecellulose whose amino acid sequence is encoded by a nucleic acidsequence from a wild-type fungus of the genus Chrysosporium.
 25. Thecomposition of claim 24, wherein the fungus is selected from the groupconsisting of Chrysosporium lucknowense, Chrysosporium lobatum,Chrysosporium merdarium, and Chrysosporium tropicum.
 26. The compositionof claim 25, wherein the fungus is Chrysosporium luckowense.
 27. Acomposition for the enzymatic treatment of cellulose fibers or fabrics,having about 120 to 191 units of endo-1,4-β-glucanase activity per gramof dry composition, as measured by an endoviscometric assay, of aneutral and/or alkaline cellulose isolated or obtained from a wild-typefungus of the genus Chrysosporium.
 28. The composition of claim 27,wherein the fungus is selected from the group consisting ofChrysosporium luckowense, Chrysosporium lobatum, Chrysosporiummerdarium, and Chrysosporium tropicum.
 29. The composition of claim 28,wherein the fungus is Chrysosporium luckowense.
 30. A laundry detergentcomposition, comprising a cellulase whose amino acid sequence is encodedby a nucleic aid sequence from a wild-type fungus of the genusChrysosporium, further comprising a surfactant.
 31. The composition ofclaim 30, wherein the fungus is selected from the group consisting ofChrysosporium lucknowense, Chrysosporium pannorum, Chrysosporiumpruinosum, Chrysosporium keratinophilum, Chrysosporium lobatum,Chrysosporium merdarium, Chrysosporium queenslandicum, and Chrysosporiumtropicum.
 32. The composition of claim 31, wherein the fungus isChrysosporium lucknownense.
 33. A laundry detergent composition,comprising a cellulase isolated or obtained from a wild-type fungus ofthe genus Chrysosporium, further comprising a surfactant.
 34. Thecomposition of claim 33, wherein the fungus is selected from the groupconsisting of Chrysosporium lucknowense, Chrysosporium pannorum,Chrysosporium pruinosum, Chrysosporium, keratinophilum, Chrysosporiumlobatum, Chrysosporium merdarium, Chrysosporium queenslandicum, andChrysosporium tropicum.
 35. The composition of claim 34, wherein thefungus is Chrysosporium luckowense.
 36. A neutral/alkaline cellulasecomposition obtained from a wild-type fungus of the speciesChrysosporium luckowense.
 37. A neutral/alkaline cellulase compositionobtained from a wild-type fungus of the species Chrysosporiumlucknowense Garg 27K, accession number VKM F-3500D.
 38. Aneutral/alkaline cellulase composition obtained from a wild-type fungusof the genus Chrysosporium, with the proviso that the fungus is notChrysosporium pruinosum.
 39. A composition for the enzymatic treatmentof cellulosic fibers or fabrics, having from 314 to 1537 units ofendo-1,4-β-glucanase activity per gram of protein, as measured by anendoviscometric assay, of a neutral and/or alkaline cellulose whoseamino acid sequence is encodes by a nucleic acid sequence from awild-type fungus of the genus Chrysosporium.
 40. The composition ofclaim 39, wherein the fungus is selected from the group consisting ofChrysosporium lucknowense, Chrysosporium lobatum, Chrysosporiummerdarium, and Chrysosporium tropicum.
 41. The composition of claim 40,wherein the fungus is Chrysosporium luckowense.
 42. A composition forthe enzymatic treatment of cellulosic fibers or fabrics, having from 314to 1537 units of endo-1,4-β-glucanase activity per gram of protein, asmeasured by an endoviscometric assay, of a neutral and/or alkalinecellulose isolated or obtained from a wild-type fungus of the genusChrysosporium.
 43. The composition of claim 42, wherein the fungus isselected from the group consisting of Chrysosporium luckowense,Chrysosporium lobatum, Chrysosporium merdarium, and Chrysosporiumtropicum.
 44. The composition of claim 43, wherein the fungus isChrysosporium luckowense.